Micro-CT for saw mark analysis on human bone.

In toolmark analysis, microscopy techniques, such as micro-CT, are used to visualise and measure toolmarks left on bones by a tool. In dismemberment cases, properties such as the width of the saw mark can provide cues to which tool was used by the culprit. The aim of the current study was to establish whether; (i) micro-CT is an appropriate imaging technique for saw mark analysis, (ii) toolmarks statistically differ when created with different tools, (iii) toolmark width can predict tool blade width, and (iv) toolmarks differ if created under different methodological conditions. Across two experiments, 270 saw marks were created using eight tools with either a controlled or free saw action on either fleshed or defleshed human long bone. Toolmarks were micro-CT scanned and seven toolmark properties were categorised or measured by two independent raters. The current study found that; (i) micro-CT was found to be a powerful and reliable imaging method for the visualisation and measurement of saw mark properties, (ii) toolmark properties differed significantly within and between various methodological conditions (p<.001) when created by eight different tools, (iii) a regression model developed using toolmark widths from Experiment 2 overall predicted 94% of tool widths in Experiment 1, and iv) methodological factors such as tissue presence and saw action significantly and inconsistently influenced toolmark properties for different tools. The study further validates the use of mirco-CT for saw mark analysis and demonstrates the potential of using toolmark properties to determine the tool used in cases of dismemberment. Given the effects that methodological factors such as tissue presence can have on toolmark properties, future studies should use experimental set ups with fleshed human tissue and use a free saw action.

The cutting edge - Micro-CT for quantitative toolmark analysis of sharp force trauma to bone.

Toolmark analysis involves examining marks created on an object to identify the likely tool responsible for creating those marks (e.g., a knife). Although a potentially powerful forensic tool, knife mark analysis is still in its infancy and the validation of imaging techniques as well as quantitative approaches is ongoing. This study builds on previous work by simulating real-world stabbings experimentally and statistically exploring quantitative toolmark properties, such as cut mark angle captured by micro-CT imaging, to predict the knife responsible. In Experiment 1 a mechanical stab rig and two knives were used to create 14 knife cut marks on dry pig ribs. The toolmarks were laser and micro-CT scanned to allow for quantitative measurements of numerous toolmark properties. The findings from Experiment 1 demonstrated that both knives produced statistically different cut mark widths, wall angle and shapes. Experiment 2 examined knife marks created on fleshed pig torsos with conditions designed to better simulate real-world stabbings. Eight knives were used to generate 64 incision cut marks that were also micro-CT scanned. Statistical exploration of these cut marks suggested that knife type, serrated or plain, can be predicted from cut mark width and wall angle. Preliminary results suggest that knives type can be predicted from cut mark width, and that knife edge thickness correlates with cut mark width. An additional 16 cut marks walls were imaged for striation marks using scanning electron microscopy with results suggesting that this approach might not be useful for knife mark analysis. Results also indicated that observer judgements of cut mark shape were more consistent when rated from micro-CT images than light microscopy images. The potential to combine micro-CT data, medical grade CT data and photographs to develop highly realistic virtual models for visualisation and 3D printing is also demonstrated. This is the first study to statistically explore simulated real-world knife marks imaged by micro-CT to demonstrate the potential of quantitative approaches in knife mark analysis. Findings and methods presented in this study are relevant to both forensic toolmark researchers as well as practitioners. Limitations of the experimental methodologies and imaging techniques are discussed, and further work is recommended.

The use of the Rx spin label in orientation measurement on proteins, by EPR.

The bipedal spin label Rx is more restricted in its conformation and dynamics than its monopodal counterpart R1. To systematically investigate the utility of the Rx label, we have attempted to comprehensively survey the attachment of Rx to protein secondary structures. We have examined the formation, structure and dynamics of the spin label in relation to the underlying protein in order to determine feasibility and optimum conditions for distance and orientation measurement by pulsed EPR. The labeled proteins have been studied using molecular dynamics, CW EPR, pulsed EPR distance measurement at X-band and orientation measurement at W-band. The utility of different modes and positions of attachment have been compared and contrasted.

The orientation of a tandem POTRA domain pair, of the beta-barrel assembly protein BamA, determined by PELDOR spectroscopy.

The outer membrane beta-barrel trans-membrane proteins in gram-negative bacteria are folded into the membrane with the aid of polypeptide transport-associated (POTRA) domains. These domains occur, and probably function, as a tandem array situated on the periplasmic side of the outer membrane. Two crystal structures and one NMR study have attempted to define the structure and articulation of the POTRA domains of the Escherichia coli, prototypic Omp85 protein BamA. We have used pulsed electron paramagnetic resonance (EPR) to determine the distance and distance distribution between (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSSL), placed across the domain interface of the first two POTRA domains of BamA. Our results show tightly defined interdomain distance distributions that indicate a well-defined domain orientation. Examination of the known structures revealed that none of them fitted the EPR data. A combination of EPR and NMR data was used to generate converged structures with defined domain-domain orientation.

The structure and active site of the Varkud satellite ribozyme.

The Varkud satellite ribozyme is the largest of the small nucleolytic ribozymes, and the only one for which there is no crystal structure. It can be divided into a trans -acting ribozyme, consisting of five helices organized by two three-way helical junctions, and a stem-loop substrate with which it interacts, primarily by tertiary interactions. We have determined the global fold of the ribozyme, and the manner by which it interacts with the substrate. The substrate interacts with a cleft formed between helices II and VI (organized by the lower helical junction), where it contacts the A730 loop, the probable active site of the ribozyme. Within this loop, there is a critical adenine base (A756) that is a candidate for direct nucleobase participation in the cleavage reaction.

The A730 loop is an important component of the active site of the VS ribozyme.

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR.

We have used (19)F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of (19)F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg(2+). The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg(2+) with an association constant in the range of 100 to 500 M(-1), depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Structure, folding and activity of the VS ribozyme: importance of the 2-3-6 helical junction.

The VS nucleolytic ribozyme has a core comprising five helices organized by two three-way junctions. The ribozyme can act in trans on a hairpin-loop substrate, with which it interacts via tertiary contacts. We have determined that one of the junctions (2-3-6) undergoes two-stage ion-dependent folding into a stable conformation, and have determined the global structure of the folded junction using long-range distance restraints derived from fluorescence resonance energy transfer. A number of sequence variants in the junction are severely impaired in ribozyme cleavage, and there is good correlation between changes in activity and alteration in the folding of junction 2-3-6. These studies point to a special importance of G and A nucleotides immediately adjacent to helix II, and comparison with a similar junction of known structure indicates that this could adopt a guanine-wedge structure. We propose that the 2-3-6 junction organizes important aspects of the structure of the ribozyme to facilitate productive association with the substrate, and suggest that this results in an interaction between the substrate and the A730 loop to create the active complex.

A conserved nuclease domain in the archaeal Holliday junction resolving enzyme Hjc.

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3' to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.

Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies.

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.

Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster.

Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases delta and epsilon, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209(B1) and mus209(2735)) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209(B1) is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209(B1) and mus209(2735) interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209(B1) and mus209(2735) homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

The Holliday junction is finally seen with crystal clarity.

After a long wait, we finally see the structure of the Holliday junction of genetic recombination at atomic resolution. It comprises a right-handed cross of DNA molecules, with an antiparallel orientation of strands.

Binding of U1A protein to the 3' untranslated region of its pre-mRNA.

We have studied the global structure of the U1A 3' untranslated region (UTR) element using fluorescence resonance energy transfer. Comparison of a single UTR-box with a series of oligoadenine bulges indicates that the UTR-box introduces a significant kink into the axis of the RNA, and quantification of the results suggests an included bend angle of approximately 100 degrees (i.e. 80 degrees from linear). The complete 3'-UTR element is also severely kinked by the two UTR-boxes. We can observe binding of U1A protein to the 3'-UTR element by a change in the fluorescence anisotropy of Cy3 attached to one of the helical ends. In parallel with the binding, we observe a marked increase in fluoresence resonance energy transfer efficiency between fluorophores attached at the two 5' termini, indicating a significant change in global conformation induced by the binding of the protein.

Molecular chaperones: small heat shock proteins in the limelight.

Small heat shock proteins have been the Cinderellas of the molecular chaperone world, but now the crystal structure of a small heat shock protein has been solved and mutation of two human homologues implicated in genetic disease. Intermediate filaments appear to be one of the key targets of their chaperone activity.

HMG box proteins bind to four-way DNA junctions in their open conformation.

The HMG box is an 80 amino acid domain found in a variety of eukaryotic chromosomal proteins and transcription factors. Binding to DNA is associated with recognition of structural distortion or manipulation of DNA structure. All the HMG box domains bind to four-way DNA junctions, which must therefore present some feature that is common to the binding targets of this wide variety of proteins. Since the four-way junction can itself adopt a variety of structures depending upon conditions, it is important to determine in which form it exists in complexes with HMG boxes. We find that a single HMG box domain is bound exclusively to the open square form of the junction and that conditions that stabilize the stacked X structure significantly lower affinity for the HMG box. We suggest that the HMG domain binds to one arm of the junction in the minor groove at the point of strand exchange and we present a model for the structure of the complex.

Severe axial bending of RNA induced by the U1A binding element present in the 3' untranslated region of the U1A mRNA.

The 3' untranslated region of the U1A mRNA contains a binding site for the U1A protein that consists of two asymmetric internal bulges. The bulges each comprise a loop of seven unpaired bases opposing a single base (termed a U1A box). The seven-base loops are located on opposite strands, distributed in a symmetrical manner about the intervening four-base duplex. We have investigated the global conformation of this binding element. Comparison of electrophoretic mobilities of RNA duplexes interrupted by a single U1A box with a series of duplexes of the same length containing oligoadenine bulges indicates that the individual boxes cause a substantial kinking of the helix axis, estimated to be 90 (+/- 10) degrees. A series of RNA duplexes were constructed containing a U1A box separated from an A5 bulge by a duplex section of length between 3 and 21 bp. It was found that the electrophoretic mobilities of these species varied sinusoidally, indicating that the U1A box introduces a defined kink into the RNA helix, rather than a point of flexibility. Electrophoretic experiments with the complete U1A binding element suggest that the axial trajectories of the two U1A boxes combine to give an approximately in-line, 180 degrees change in duplex direction.

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

hSRY: molecular gender bender.

The recently published solution structure of the DNA-binding domain of hSRY in complex with a DNA octamer offers insight into the sex reversal effects of mutations in hSRY and the interaction of all HMG boxes with DNA.

Characterisation of the interaction between PCNA and Gadd45.

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.

Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata B1.

The amino acid sequence and structure of a uterotonic polypeptide extracted from the African plant Oldenlandia affinis DC have been determined. The peptide, kalata B1, consists of 29 amino acid residues and is rich in cysteine (6), threonine (5), and glycine (5). Enzyme cleavage studies show that the polypeptide backbone is cyclic. The three-dimensional solution structure has been determined using two-dimensional nuclear magnetic resonance (NMR) spectroscopy and distance-restrained simulated annealing. Kalata B1 is composed mainly of beta-strands connected by tight turns, forming regions of beta-sheet, except in the case of one section which forms a longer, less structured loop. The tertiary fold, together with the disulfides that form a sulfur core, produces a striking and unusual surface in which the majority of the hydrophobic residues form a solvent-exposed patch. The hydrophobic side of kalata B1 is flanked by two diametrically opposed and opposite-charged residues. The structure calculations have been used to predict the previously unknown disulfide bond connectivities using two approaches. In the first, a family of structures was calculated on the basis of NOE constraints without the assumption of a specific disulfide connectivity. The resultant structures were examined to determine whether the calculated position of the sulfur atoms suggested that one set of disulfide connectivities was more likely than the other, theoretically possible, sets. In the second approach, a separate family of structures (50 per set) was calculated for each of the 15 possible disulfide-bonded molecules. The resultant families of structures were compared to see whether one was favored over the others. Both approaches led to the same global fold, and the most likely disulfide connectivity is predicted to be 5-22, 13-27, and 17-29. In the calculated structure the cyclic peptide backbone is folded back onto itself and braced with disulfide pairs across diagonally opposed beta-strands. This structure involves one of the disulfide bonds (5-22) threading through the eight amino acid loop formed by the other two disulfide bonds and the peptide fragments connecting them.