Biomedical subjects

A P Wiles

Publications and source records attributed to A P Wiles.

3 recordsLinked to original sources

PubMed1999

The human eukaryotic initiation factor 4AI gene (EIF4A1) contains multiple regulatory elements that direct high-level reporter gene expression in mammalian cell lines.

The gene encoding human eukaryotic initiation factor 4A (EIF4A1) is located on chromosome 17p13, 667 bp upstream from the gene encoding the macrophage endosomal protein CD68. The EIF4AI gene contains 10 intervening sequences with the 1397-bp first intron containing a CpG-rich methylation-free island. Sequences capable of enhancing gene expression reside between positions -69 and -371 and positions -504 and -1100 of the EIF4AI 5' flanking sequence and within introns 1, 2, 3, 7, and 9. In macrophage cell lines, EIF4A1 expression vectors give sustained high-level reporter gene expression to levels 10 times higher than that obtained using the human cytomegalovirus immediate-early gene promoter/enhancer. Sequences of the human EIF4AI gene may find application in the development of new vectors for gene therapy and genetic vaccination.

5' Untranslated Regions↗

PubMed1997

NMR studies of a viral protein that mimics the regulators of complement activation.

Vaccinia virus complement control protein (VCP) is a 243-residue protein that is similar in sequence to the regulators of complement activation; its role is to defend the virus against attack by the host complement system. A fragment of this protein spanning the two complement protein (CP)-modules (residues 126 to 243) which make up the C-terminal half of VCP has been expressed in Pichia pastoris. A 15N-labelled sample was purified for the purposes of structure determination and measurements of dynamics in solution using NMR. Structures were calculated on the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence. The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; average r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons,=1.2 A) but less similar to the fifth CP-module of fH (average r.m.s.d.=2.2 A). In the VCP fragment, the orientation of one module with respect to the other is clearly defined by the experimental data, and T1 measurements are consistent with only limited flexibility at the module-module interface. The r.m.s.d. over all of the 118 residues (backbone atoms) is 0.73 A. The intermodular orientation is better defined than, and significantly different from, that observed in a CP-module pair from fH (re-calculated using the extended SA protocol). In VCP the long axis of the second module is tilted by 59(+/-4) degrees with respect to the first module (50(+/-13) degrees in the fH pair), and twisted with respect to the first module by 22(+/-6) degrees (223(+/-17) degrees in fH). The differences between the human and viral proteins may be rationalised in terms of the lack of hydrogen-bond stabilised secondary structure in the N-terminal portion of fH module 16, and the number and type of amino acid side-chains which make up the interface. A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matrix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimental data.

Amino Acid Sequence↗

PubMed1993

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Amino Acid Sequence↗