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J Prosser

Publications and source records attributed to J Prosser.

At least 37 records · Page 2Linked to original sources
PubMed1994

APC mutation analysis by chemical cleavage of mismatch and a protein truncation assay in familial adenomatous polyposis.

Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) who have been included in studies reported in the literature. In order to determine the true frequency of detectable APC mutations, we set out to search exhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be linked to 5q markers. Chemical cleavage of mismatch analysis was employed as the initial screening technique. Mutations were confirmed by direct DNA sequencing and shown to generate a premature stop codon by an in vitro protein synthesis assay. Mutations resulting in a premature stop codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intragenic APC markers with a lodscore of 1.69 at Zmax = 0.0, further analysis of DNA, RNA and chromosome spreads from the proband failed to detect any abnormality. This was despite employing single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, reverse transcription-polymerase chain reaction (RT-PCR) analysis for splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (FISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the numerous other techniques employed could detect the mutation in the remaining kindred. This study shows the value of screening the APC gene using a combination of chemical cleavage of mismatch analysis and an in vitro protein truncation test.

Adenomatous Polyposis Coli↗
PubMed1993

Detection of point mutations in the p53 gene: comparison of single-strand conformation polymorphism, constant denaturant gel electrophoresis, and hydroxylamine and osmium tetroxide techniques.

A comparison was made between the 3 most commonly used techniques for the detection of point mutations: single-strand conformation polymorphism (SSCP), constant denaturant gel electrophoresis (CDGE), and hydroxylamine and osmium tetroxide used in amplification mismatch cleavage analysis (HOT). Using human DNA samples containing known mutations in the p53 gene, SSCP detected 90% of mutations (18/20), CDGE detected 88% (15/17) pre-decoding of the samples but 100% when the mutations were known and the CDGE conditions optimized, and the HOT technique was 100% accurate, although 1 mutation was missed through careless examination of the gel. The positive and negative aspects of each of the techniques are considered and suggestions are made regarding the particular situations in which each of them is most useful.

DNA, Single-Stranded↗
PubMed1993

Detecting single-base mutations.

The ability to detect single-base changes is of fundamental importance in molecular genetics. This is particularly true in human genetics, where interest in linking mutations of identified genes to particular disease phenotypes is most intense, and where a demand exists for clinical diagnosis of defined mutations. In the following article, techniques are described for screening unknown mutations, as well as diagnosing those that have been identified previously. The underlying methods are explained briefly and guidelines are offered for choosing one technique in preference to another.

Animals↗
PubMed1992

p53 mutations in breast cancer.

We have identified and analyzed 41 mutations in p53 in sporadic breast tumors from 136 unselected breast cancer patients and estimate that approximately 40% of such tumors contain p53 mutations. The frequency of G-T transversions and the incidence of guanosine mutations in the nontranscribed strand of the p53 gene were found to be higher than expected, and we suggest, therefore, that exogenous carcinogens have an etiological role in sporadic breast cancers. Mutations were recorded in 44 codons of the p53 gene, with no obvious mutational hot-spots, although mutations at codons 175, 194, 273, and 280 accounted for 25% of the changes. One germ-line mutation was found in 136 patients and so we conclude that constitutional mutation of p53 may be an uncommon etiological factor in breast cancer.

Base Composition↗
PubMed1992

Zinc finger point mutations within the WT1 gene in Wilms tumor patients.

A proposed Wilms tumor gene, WT1, which encodes a zinc finger protein, has previously been isolated from human chromosome 11p13. Chemical mismatch cleavage analysis was used to identify point mutations in the zinc finger region of this gene in a series of 32 Wilms tumors. Two exonic single base changes were detected. In zinc finger 3 of a bilateral Wilms tumor patient, a constitutional de novo C----T base change was found changing an arginine to a stop codon. One tumor from this patient showed allele loss leading to 11p hemizygosity of the abnormal allele. In zinc finger 2 of a sporadic Wilms tumor patient, a C----T base change resulted in an arginine to cysteine amino acid change. To our knowledge, a WT1 gene missense mutation has not been detected previously in a Wilms tumor. By comparison with a recent NMR and x-ray crystallographic analysis of an analogous zinc finger gene, early growth response gene 1 (EGR1), this amino acid change in WT1 occurs at a residue predicted to be critical for DNA binding capacity and site specificity. The detection of one nonsense point mutation and one missense WT1 gene point mutation adds to the accumulating evidence implicating this gene in a proportion of Wilms tumor patients.

Amino Acid Sequence↗
PubMed1992

p53 allele losses, mutations and expression in breast cancer and their relationship to clinico-pathological parameters.

The p53 locus on the short arm of chromosome 17 at 17p 13.1 was examined for loss of heterozygosity, mutation, mRNA and protein expression in 60 primary breast cancers. Allele loss around the p53 locus was detected in 19/45 informative tumours (42%). p53 mutations in the evolutionarily conserved exons 5 to 9 were detected in 17/60 (28%) by amplification mismatch and confirmed by direct DNA sequencing. p53 mRNA expression was detected by Northern blot in 36/59 (61%) of tumours, and p53 protein expression using antibody 1801 on frozen-tissue sections in 13/44 of the tumours examined. p53 mutation was significantly associated with oestrogen-receptor-poor tumours (p less than 0.01) and hence with poor prognosis, but not with other clinical or pathological parameters. There was no statistical correlation between loss of heterozygosity around the p53 locus at 17p13.1 and p53 mutation. Furthermore, p53 mutation was not associated with p53 expression detected by immunohistochemical staining with antibody 1801 or as p53 mRNA. In addition, events on 17p (allele losses, p53 mutation, p53 expression) were independent of c-erbB-2 expression. In breast cancer, by contrast with colorectal, lung and ovarian cancer, there appears to be no clear association between p53 DNA abnormalities and p53 expression.

Alleles↗
PubMed1992

The human PAX6 gene is mutated in two patients with aniridia.

Aniridia is an inherited ocular disorder of variable expressivity characterized by iris hypoplasia. A candidate aniridia gene, AN, which is the human homologue of the mouse Pax-6 gene, has recently been isolated by positional cloning from the WAGR region of 11p13. Here we describe mutations in this gene in two cases of sporadic aniridia, one detected at the DNA level and one at the RNA level, both of which are predicted to affect protein function. Mutations in Pax-6 have been described previously in Small eye, the proposed mouse model for aniridia. We present new phenotypic evidence for the validity of this mouse model.

Animals↗
PubMed1992

Tumor-suppressor genes: cardinal factors in inherited predisposition to human cancers.

A predisposition to the development of certain specific and familial cancers is associated with the inheritance of a single mutated gene. In the best-characterized cases, this primary mutation is a loss of function mutation consistent with viability but resulting in neoplastic change consequent to the acquisition of a second somatic mutation at the same locus. Such genes are referred to as tumor-suppressor genes. Classical examples are the Rb-1 gene associated with the development of retinoblastoma and the p53 gene, which is associated with a wider range of neoplasms, including breast cancer. Other tumor-suppressor genes have been isolated which are associated with Wilms' tumor, neurofibromatosis, and inherited and sporadic forms of colorectal cancer. Some of these genes appear to act as negative regulators of mitotic cycle genes, and others may have different properties. The nature of these genes is discussed, as is the evidence for the involvement of tumor-suppressor genes in other inherited, and sporadic, forms of cancer. Some recent data on the Wilms' tumor gene, WT1, and on the involvement of the p53 gene in breast cancer are presented, and the importance of genomic imprinting in contributing to the excess of suppressor gene mutations in chromosomes of paternal origin is considered.

Breast Neoplasms↗
PubMed1991

A familial RB1 mutation detected by the HOT technique is homozygous in a second primary neoplasm.

Using the hydroxylamine-osmium tetroxide (HOT) technique, we have identified a constitutional point mutation in the retinoblastoma susceptibility gene (RB1) which segregates with the expression of retinoblastoma in five affected family members. One member developed a second primary tumor, a small-cell lung carcinoma (SCLC), which metastasized to the liver. Analysis of liver tumour DNA revealed homozygosity for the constitutional mutation, a G----A transition at the fifth base of intron 21, resulting in the excision of exon 21 from the mRNA. This is the first demonstration of homozygotization of a constitutional RB mutation in a metastatic second primary tumour and underlines the usefulness of the HOT technique for identification of mutations of the RB1 gene.

Adult↗
PubMed1990

Identification of a potential RNA intermediate for transposition of the LINE-like element I factor in Drosophila melanogaster.

The I factor, a transposable element related to mammalian LINEs, controls the I-R system of hybrid dysgenesis in Drosophila melanogaster. It transposes at high frequency in the germ-line of the female progeny of crosses between females of the reactive class of strains and males of the inducer class. The structure and DNA sequence of the I factor suggest that it transposes by reverse transcription of an RNA intermediate. Northern blot and S1 mapping experiments show that a full-length RNA of the I factor is synthesized specifically in the conditions of which I factors transpose. This RNA has all characteristics of a transposition intermediate. It is only found in the ovaries of dysgenic females suggesting that I factor activity is restricted to this tissue because of regulation at the level of the initiation of transcription or RNA stability.

Animals↗
PubMed1990

Evidence that p53 behaves as a tumour suppressor gene in sporadic breast tumours.

Using the polymerase chain reaction, we have amplified exons 5 and 6 of the human p53 gene from paired samples of tumour and blood DNA of 60 patients with sporadic breast tumours and from placental or tonsil DNA of 30 normal controls. The patients' DNAs were previously examined for loss of heterozygosity of markers distal to the p53 gene on chromosome 17p and tumour samples were assayed for p53 mRNA levels. Hydroxylamine modification of mismatched base pairs was used to identify mutations in the amplified product. We have directly sequenced many of the samples including all those with mutations and have identified the particular mutation in each case. Exons 5 and 6 code for amino acids 126 through to 224 and constitute approximately 25% of the total coding region of the gene. They encompass part of the SV40 binding region as well as 2 of 5 areas known to be highly conserved in evolution and mutations in this region have been shown to be associated with tumorigenicity. We have found mutations in 13% of the primary tumours (8/60), all of which show allele loss of markers in the 17p region. No mutations were found in exons 5 and 6 of 30 normal controls. Our results are the first to identify mutations in p53 in primary breast tumours. Since our analysis has so far been confined to only part of the coding region of the gene, it is likely that further studies will reveal a mutation frequency in excess of 13%.

Base Sequence↗
PubMed1988

Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds.

Basic fibroblast growth factor (bFGF) is a polypeptide found within the CNS with potent effects on the survival and proliferation of CNS glia and endothelial cells, and on the survival and outgrowth of CNS neurons. Immunohistochemical methods were used to examine relative changes in the levels and distribution of bFGF following focal brain injury. Two monospecific antisera to bFGF were used to immunostain intact mature rat brain, and brain in which a focal mechanical lesion had been made in the dorsolateral cerebral cortex one week previously. In the intact brain, staining was localized primarily in neuronal cell bodies, especially in limbic structures. In injured brain, a marked increase of bFGF immunoreactivity was found at the borders of lesions, localized to the dense accumulation of cells, many of which resembled 'reactive' astroglia. Such increases in local bFGF concentrations may contribute to the cascade of cellular changes--including glial and capillary proliferation, and neural sprouting--that follows focal brain injury.

Animals↗
PubMed1988

Hydrogen-mediated mannose uptake in Azotobacter vinelandii.

Azotobacter vinelandii can grow mixotrophically with H2 plus mannose under N2-fixing conditions (T. Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). Mixotrophically grown cultures incubated in H2 transported mannose with a Vmax fourfold greater than that observed for cultures incubated in argon, but H2 did not change the apparent Km for mannose. Respiratory inhibitors, such as potassium cyanide, hydroxylamine, and p-chloromercuribenzoic acid, as well as the proton conductor carbonyl cyanide m-chlorophenyl-hydrazone inhibited mannose uptake. We suggest that one of the roles of H2 in mixotrophic metabolism is to supply energy that facilitates mannose transport.

Azotobacter↗