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D R Turner

Publications and source records attributed to D R Turner.

At least 73 records · Page 4Linked to original sources
PubMed1988

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.

Base Sequence↗
PubMed1988

Prenatal and adult presymptomatic testing for Huntington's disease.

The discovery of a fragment of DNA that is linked closely to the Huntington's-disease autosomal locus offers the opportunity for the presymptomatic diagnosis of this dominantly-inherited neurodegenerative disorder. Presymptomatic testing will present individuals and society with difficult choices and responsibilities. A pilot adult presymptomatic test programme is under way for SA families. Presymptomatic testing requires intensive counselling both before and after the test. A form of prenatal test, which is applicable to a significant proportion of couples with one partner at risk of Huntington's disease, is available also. As this form of prenatal test does not change the risk status of the parent, less extensive counselling is required and testing is available nationally through the SA programme. It is anticipated that other states will develop their own diagnostic programmes in the near future. This article explains the basis for the test, its accuracy and the importance of obtaining DNA from key individuals in pedigrees of Huntington's disease.

Female↗
PubMed1988

Chronic serum sickness glomerulonephritis: modification of the immune response influences the rate of removal of mesangial electron-dense deposits.

We have used a chronic serum sickness model of glomerulonephritis to investigate whether gross interference with the immune system can influence the rate of removal of antigen and established electron-dense deposits from the glomerulus. Radio-labelled cationized bovine serum albumin (BSA) was used as antigen. During the 2 weeks after the cessation of injections, the rate of removal of antigen from isolated glomeruli and from renal cortex, liver, spleen, and lung was measured. The rate of removal of mesangial and subepithelial deposits was assessed by point-counting. Urinary excretion of free and protein-bound isotope was also measured. Having quantified the rate of removal of antigen and deposits from the glomerulus, we attempted to influence the rate of removal by interfering with the immune response in the course of recovery. Contrary to our expectations, stimulation of the immune system with antigen in Freund's complete adjuvant, 4 days after the last injection of antigen, inhibited the removal of antigen and mesangial electron-dense deposits. Prednisolone had no detectable effect, but large doses of a non-nephritogenic form of the antigen (native BSA) enhanced removal. Removal of antigen and mesangial deposits was inversely correlated with the levels of circulating anti-BSA antibody, suggesting that specific antibody, circulating through the mesangium, inhibits the removal of antigen which is already trapped at that site. None of the forms of intervention applied during recovery produced a detectable change in the rate of removal of subepithelial deposits.

Animals↗
PubMed1988

Chronic serum sickness glomerulonephritis: passive immunisation inhibits the removal of glomerular antigen and electron dense deposits.

Chronic serum sickness glomerulonephritis was induced in 20 Wistar rats, using radiolabelled, chemically cationized bovine serum albumin (BSA) as antigen. Four days after the last injection of antigen, when relocation of antigen within the rat had effectively ceased, the rats were given a single large intraperitoneal dose of either non-immune rat gamma globulin or anti-BSA rat gamma globulin. Ten days later the rats were killed. The rats which had received the anti-BSA globulin had significantly more antigen in renal cortex and in isolated glomeruli than the control group. They also had larger mesangial deposits as assessed by morphometry at electron microscope level; assessment of subepithelial deposits provided equivocal results. These findings provide direct confirmation that circulating antibody which is directed against an antigen which is trapped within deposits in the glomerulus will inhibit the removal of the antigen and deposits from the mesangium.

Animals↗
PubMed1988

Mutations in human lymphocytes studied by an HLA selection system.

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.

Alleles↗
PubMed1988

Karyotypic abnormality of the X chromosome is rare in mutant HPRT-lymphocyte clones.

Lymphocyte clones mutated at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus on the X chromosome were studied by synchronization and G banding to determine the proportion of mutant clones having visible karyotypic change. 47 spontaneously mutant clones, 17 mutant clones induced by X-irradiation and 33 wild-type clones were studied. All clones were karyotypically normal except for 1 clone induced by X-irradiation in which an interstitial deletion of the short arm of the X chromosome had been inserted into the long arm of the same chromosome between q23 and q24; this change may have been coincidental or may have resulted in a position effect mutation. It was concluded that the great majority of mutations were not associated with a visible chromosome abnormality. This conclusion complements molecular studies which suggest that gene changes at the HPRT locus in HPRT- mutants generally extend over segments of DNA too small to be resolved by karyotypic analysis.

Cells, Cultured↗
PubMed1988

Mutations in human lymphocytes commonly involve gene duplication and resemble those seen in cancer cels.

Mutations in human lymphocytes are commonly due to gene deletion. To investigate the mechanism of deletion for autosomal genes, we immunoselected lymphocytes mutated at the HLA-A locus and cloned them for molecular analysis. Of 36 mutant clones that showed deletion of the selected HLA-A allele, 8 had resulted from a simple gene deletion, whereas 28 had resulted from a more complex mutational event involving reduplication of the nonselected HLA-A allele as indicated by hybridization intensity on Southern blots. In 3 of the 28 clones, retention of heterozygosity at the HLA-B locus indicated that the reduplication was due to recombination between the two chromosomes 6; but in the remaining 25 clones, distinction could not be made between recombination and chromosome reduplication. The results indicate that mutations in normal somatic cells frequently result in hemizygosity or homozygosity at gene loci and, thereby, resemble the mutations thought to be important in the etiology of various forms of cancer.

Alleles↗
PubMed1988

Chronic serum sickness glomerulonephritis: removal of glomerular antigen and electron-dense deposits is largely dependent on plasma complement.

Chronic serum sickness glomerulonephritis was induced in 20 rats, using radio-labelled cationised bovine serum albumin (BSA) as antigen. Four days after the last dose of antigen, half the rats were given cobra venom factor (CVF) in doses sufficient to render plasma complement activity undetectable. After ten days without circulating complement, the rats had significantly more antigen in glomeruli than the control group. Subepithelial and mesangial electron dense deposits are found in this model: morphometric analysis indicated that the elimination of plasma complement had slowed the removal of deposits at both of these sites. A second experiment studied the kinetics of this effect. CVF was again given four days after the last dose of antigen, but rats were killed in small groups at intervals thereafter. The results indicate that initially decomplementation had little effect, but after seven days without complement the removal of glomerular antigen had virtually ceased. The complement depleted rats had significantly lower levels of proteinuria, despite having more antigen and larger deposits in their glomeruli. In this model therefore, complement appears to be essential for the removal of deposits, and simultaneously contributes significantly to the induction of proteinuria.

Animals↗
PubMed1987

Immune complex-induced defects in glomerular basement membrane charge can be "repaired" despite continuing glomerular disease.

There have been conflicting reports over whether or not glomerular immune complex deposits cause proteinuria by neutralizing the glomerular basement membrane anionic charge. We investigated whether the reported discrepancies could arise as a result of repair of the charge layer, in the face of continuing antigen deposition, and how rapidly this repair could be effected. We used a chronic serum sickness model of glomerulonephritis which consistently produces a nephrotic syndrome stable over 8 weeks. Charge distribution was demonstrated by the polyethyleneimine (PEI) method. Subepithelial electron dense deposits were associated with defects in the charge layer of the lamina rara externa at 2 and 4 weeks, but at 8 weeks many of the deposits had become surrounded by a new charge layer. This finding is of relevance to the interpretation of similar studies on human material.

Animals↗
PubMed1987

Effect of age on lymphocyte proliferation.

The relationship of lymphocyte proliferative capacity to age was studied using lymphocytes from neonates, from young adults aged 20-30 and from healthy individuals aged 70-90. Mass cultures expanded exponentially and eventually died after a final expansion of 10(19)-10(52). They therefore showed a Hayflick effect, but in contrast to reported findings for other cell types there was no relationship between age and the magnitude of the final expansion. Cytogenetic and molecular studies showed that monoclonality developed in all mass cultures. Study of individual clones also showed exponential growth followed by cessation. The magnitude of the expansion, 10(4)-10(35), was substantially less than that observed for mass cultures, but was related to age. We conclude that lymphocytes have a heterogeneous proliferative potential, that the overall proliferative potential declines with age but that rare cells having extended proliferative potential continue to be present into old age. The development of monoclonality during the course of mass cultures has implications for the interpretation of findings from such cultures since observations drawn from the later stages of culture reflect the properties of rare cells having high proliferative potential and do not necessarily reflect the properties of the overall population.

Adult↗
PubMed1987

Diagnostic use of immunoglobulin and T-cell receptor gene rearrangements in lymphoproliferative disease.

Gene rearrangements of immunoglobulin and T cell antigen receptor gene loci were studied in 67 patients referred to a general hematology clinic. The results of gene analysis supported the clinical diagnosis in most cases where involved tissue was studied. However, in a number of cases, gene rearrangement studies gave unexpected results of possible diagnostic significance. Thus two patients who presented diagnostic difficulties were finally diagnosed as having non-Hodgkin's lymphoma; gene analysis suggested that the initial diagnosis of Hodgkin's disease may have been correct. In two patients who had chronic lymphocytosis and were suspected of having chronic lymphocytic leukemia, no evidence of monoclonal disease of B or T cells could be found and the basis for the lymphocytosis was presumed to be non-malignant. Gene analysis was able to detect monoclonal B cell disease in the otherwise normal blood of two of six patients with lymphoma, one of two patients with macroglobulinemia, and none of six patients with myeloma. The study of gene rearrangements may be practical aid to diagnosis in some situations of uncertainty.

Humans↗
PubMed1987

An assessment of the influence of antigen dose in two new models of chronic serum sickness glomerulonephritis in the rat.

Two new models of chronic serum sickness glomerulonephritis have been developed and characterized, using cationic and native bovine serum albumin (BSA). During this development, it has become apparent that there exists an optimum nephritogenic dose for native (anionic) BSA, above which the severity of glomerular changes diminishes; but for cationic BSA, higher doses consistently produce more severe lesions. This finding supports the theory that antigens of different charge are deposited in the glomerulus by different mechanisms. We have also found that cationic BSA circulates not in the blood plasma, but mainly bound to red cells. The two experimental models have proved to be more convenient and more consistent than those previously reported; the cationic BSA model also induces heavy proteinuria and the nephrotic syndrome. They will facilitate further studies of how antigen-antibody complexes are handled by the glomerulus in chronic immune complex disease.

Animals↗
PubMed1986

Essential features of the assembly origin of tobacco mosaic virus RNA as studied by directed mutagenesis.

The assembly origin of tobacco mosaic virus RNA contains three stable hairpin loops. Coat protein disks bind first to loop 1 (the 3' most) during virus assembly, but the whole region is coated in a concerted fashion even in conditions of limiting protein. It is shown by in vitro packaging assays using mutant assembly origin transcripts that rapid and specific assembly initiation occurs in the absence of loops 2 and 3, but is abolished on removal of loop 1. Deletion or alteration of the unpaired AAGAAGUCG sequence at the apex of loop 1 also abolishes rapid packaging; this sequence is therefore instrumental in disk binding. Alteration of this sequence to (A)9 leads to packaging at a very low rate (half time 12 hours) which is apparently non-sequence specific. Substitution of (CCG)3 evokes packaging with a half time of 3 hours, as compared to 15 seconds for the wild type assembly origin. These results suggest that the three-base G periodicity within this sequence element is an important feature in assembly nucleation.

Base Sequence↗
PubMed1986

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly.

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.

Base Sequence↗
PubMed1986

Basement membrane charge in human glomerular disease.

Fixed anionic charges in the mammalian glomerulus, on both the basement membrane and the epithelial cell foot processes, are believed to form an important part of the glomerular filtration barrier. There is good evidence that their loss causes proteinuria. The charges can be visualized ultrastructurally using cationic dyes, but the requirement of these techniques for perfusion or immersion of fresh tissue has largely confined such studies to experimental models. We have extended the widely used polyethyleneimine technique, to study the charge of glomerular basement membranes in human tissue reprocessed out of paraffin blocks up to 10 years old. We studied selected cases of glomerular disease, where the diagnosis was not in any doubt. In the majority of diseases studied, a continuous charge layer persisted despite severe abnormalities of the basement membrane. Two exceptions were found. In amyloidosis, accumulation of fibrils was associated with a considerable decrease or loss of stainable basement membrane charge. In S.L.E., numerous small defects in the charge layer were noted. The persistence of charge is contrary to reported findings in several animal models of glomerular disease, including puromycin nephrosis, Heymann nephritis and streptozotocin diabetes. Although this method is not subject to precise quantitative analysis, we conclude that in the majority of cases, proteinuria in man is not caused by an extensive loss of glomerular basement membrane anionic charge.

Anions↗