Biomedical subjects

A F Markham

Publications and source records attributed to A F Markham.

At least 19 recordsLinked to original sources

PubMed2000

Promoter analysis of the human ubiquitin-conjugating enzyme gene family UBE2L1-4, including UBE2L3 which encodes UbcH7.

UbcH7 is a ubiquitin-conjugating enzyme mediating c-fos degradation, transcription factor NF-kappaB maturation, human papilloma virus-mediated p53 and Myc protein degradation, in vitro. Previously, we characterised a highly dispersed gene family, UBE2L1-UBE2L4, whose members could potentially encode different isoforms of the UbcH7 protein. UBE2L3, located at chromosome 22q11.2, is the only identified family member with introns and encodes a polypeptide sequence identical to that of UbcH7. Promoter characterisation of UBE2L1, UBE2L3 and UBE2L4 5'-upstream regions was performed to establish which are transcribed under normal physiological conditions and after heat shock. Promoter activity was observed only with the UBE2L3 construct, the minimal promoter lying within a region 100 bp upstream of the transcriptional start site. No evidence for the presence of UBE2L1 or UBE2L4 transcripts was observed in human or murine tissues and cell lines. These data strongly suggest that UBE2L1 and UBE2L4 are likely to encode pseudogenes. Sequencing revealed that the UBE2L3 promoter contained no TATA or CCAAT boxes. Protein:DNA interaction studies confirmed the presence of binding sites for the transcription factors AP2 and Sp1 in the UBE2L3 minimal promoter. Deletion of these binding sites indicated that these factors are crucial for transcription of this gene.

Animals↗

PubMed2000

Regulation and function of the interaction between the APC tumour suppressor protein and EB1.

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Vertebrate pseudogenes.

Pseudogenes are commonly encountered during investigation of the genomes of a wide range of life forms. This review concentrates on vertebrate, and in particular mammalian, pseudogenes and describes their origin and subsequent evolution. Consideration is also given to pseudogenes that are transcribed and to the unusual group of genes that exist at the interface between functional genes and non-functional pseudogenes. As the sequences of different genomes are characterised, the recognition and interpretation of pseudogene sequences will become more important and have a greater impact in the field of molecular genetics.

Animals↗

PubMed2000

Analysis of the serotonin transporter gene linked polymorphism (5-HTTLPR) in anorexia nervosa.

Previous studies have demonstrated aberrant expression of serotonin in individuals with an eating disorder. Given this the serotonin transporter gene (5-HTT) is a strong candidate to contribute to the genetic component of the aetiology of eating disorders. To determine the role of this particular gene in the susceptibility to anorexia nervosa (AN) we have examined a tandemly repeated sequence close to the promotor region of the 5-HTT gene, which is represented by a long (L) and short (S) variant. Previous studies have shown that the transcriptional activity of the 5-HTT gene differs significantly between these two alleles. A group of 138 Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria AN patients and 90 controls were genotyped at the 5-HTT gene linked polymorphism (5-HTTLPR). Statistical analysis showed no significant difference in allele or genotype frequencies between the two groups. These data suggest that there is no association between 5-HTTLPR genotype and susceptibility to AN, in our population. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:53-55, 2000.

Adolescent↗

PubMed2000

Localization of cyclooxygenase-2 in human sporadic colorectal adenomas.

A putative target for the anti-colorectal cancer action of nonsteroidal anti-inflammatory drugs is the inducible isoform of cyclooxygenase (COX), COX-2. COX-2 is expressed within intestinal adenomas in murine polyposis models, but expression has been poorly characterized in human colorectal neoplasms. Therefore, we investigated the localization of the COX-2 protein in human sporadic colorectal adenomas. Immunohistochemistry for COX-2 and CD68 (a tissue macrophage marker) was performed on formalin-fixed, paraffin-embedded (n = 52) and frozen, acetone-fixed (n = 6) sections of human sporadic colorectal adenomas. Forty of 52 (77%) formalin-fixed adenomas expressed immunoreactive COX-2. COX-2 was localized to superficial interstitial macrophages in 39 cases (75%) and to deep interstitial macrophages in 9 cases (17%). COX-2 staining of dysplastic epithelial cells was observed in 15 cases (29%). A logistic regression analysis identified the adenoma site (P = 0.012) and histological type (P = 0.001) as independent predictors of superficial macrophage COX-2 expression. There was no relationship between the number of macrophages within an adenoma and macrophage COX-2 expression. These results indicate that COX-2 is expressed predominantly by interstitial macrophages within human sporadic colorectal adenomas. If COX-2 does indeed play a role in the early stages of colorectal carcinogenesis in man, these data suggest COX-2-mediated paracrine signaling between the macrophages and epithelial cells within adenomas.

Adenoma↗

PubMed2000

Prenatal diagnosis of aniridia.

OBJECTIVE: To use molecular genetic techniques to prenatally screen for aniridia. DESIGN: Case report. METHODS: DNA was extracted from cultured fibroblasts obtained through amniocentesis. Two mutation detection methods, Ava1 restriction digestion and single-strand conformational polymorphism electrophoresis, were used to screen the PAX6 gene. MAIN OUTCOME MEASURES: The results from the amniocentesis sample were compared with DNA obtained from the affected father, firstborn infant, and unaffected mother to determine whether the fetus carried the PAX6 mutation. RESULTS: DNA from the fetus demonstrated the same banding pattern as the affected father and firstborn infant. CONCLUSIONS: The fetus carried the mutated PAX6 allele and was predicted to develop aniridia. This was later confirmed when the child was born. This case report illustrates an important use of genetic mutation screening in the clinical setting.

Adult↗

PubMed2000

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

Adenomatous Polyposis Coli Protein↗

PubMed2000

A gene for ataxic cerebral palsy maps to chromosome 9p12-q12.

Cerebral palsy (CP) has an incidence of approximately 1 in 750 births, although this varies between ethnic groups. Genetic forms of the disease account for about 2% of cases in most countries, but contribute a larger proportion in certain sub-types of the condition and in populations with a large proportion of consanguineous marriages. Ataxic cerebral palsy accounts for 5-10% of all forms of CP and it is estimated that approximately 50% of ataxic cerebral palsy is inherited as an autosomal recessive trait. We have identified a complex consanguineous Asian pedigree with four children in two sibships affected with ataxic cerebral palsy and have used homozygosity mapping to map the disorder in this family. A genome-wide search was performed using 343 fluorescently labelled polymorphic markers and linkage to chromosome 9p12-q12 was demonstrated. A maximum Lod score of 3.4 was observed between the markers D9S50 and D9S167 using multipoint analysis, a region of approximately 23cM. We have identified a family that segregates both ataxic CP and ataxic diplegia and have mapped the genetic locus responsible in this family to chromosome 9p12-q12. The identification of gene(s) involved in the aetiology of CP will offer the possibility of prenatal/premarital testing to some families with children affected with the disorder and will greatly increase our understanding of the development of the control of motor function.

Alleles↗

PubMed2000

Lack of linkage between chromosome 5q23-33 markers and IgE/bronchial hyperreactivity in 67 Scottish families.

BACKGROUND: Raised serum immunoglobulin E (IgE) and bronchial hyperreactivity (BHR) are risk factors for the expression of the asthma phenotype. Previous studies have reported evidence for linkage between these traits and markers on the 5q23-33 cytokine gene cluster. OBJECTIVE: To test for linkage between total serum IgE/BHR and microsatellite markers which map to the 5q23-33 region in an ethnically distinct cohort of families from Aberdeen, Scotland. METHODS: We performed a linkage study between five polymorphic markers (spanning the chromosome 5q23-33 region) and total serum IgE and BHR traits. A cohort of 67 families, who were recruited originally to study the natural history of wheeze, were clinically characterized and genotyped for D5S404, IL4, IRF-1, IL9, D5S436 markers. Linkage analyses were performed using the nonparametric Haseman-Elston algorithm for the quantitative trait log IgE, and the nonparametric LOD score (NPL-score) of the GENEHUNTER package for the qualitative traits serum IgE and BHR. RESULTS: The results of the nonparametric linkage analysis using either the Haseman-Elston algorithm or NPL-score were consistent and showed no evidence for linkage with IgE. There was also no evidence for linkage between the BHR traits (at cut-off values of PD20FEV1 < 8 mmol and 16 mmol) and any of the tested five microsatellite markers. CONCLUSIONS: This study presents evidence against the presence of a gene with a major effect on total serum IgE or BHR in the 5q23-33 region, in this ethnic group.

Adolescent↗

PubMed2000

No polymorphism in the tissue transglutaminase gene detected in coeliac disease patients.

BACKGROUND: The autoantigen for the anti-endomysial antibody (AEA) found in coeliac disease has recently been reported to be the enzyme tissue transglutaminase (tTG). Polymorphisms in the gene for tTG would result in different enzymic isoforms being expressed. Certain isoforms may interact with gliadin to create antigenic neoepitopes, which could then generate an immune response in genetically predisposed individuals possessing major histocompatibility complex (MHC) class II DQ2. METHODS: We have sequenced the coding region of tTG in coeliac patients and normal controls. Total RNA was extracted from mucosal biopsies from eight AEA-positive histologically proven coeliac disease patients and four control patients with a histologically normal duodenum and a negative AEA. The 2-kb coding region of tTG was amplified in overlapping fragments by reverse transcription polymerase chain reaction (PCR), using five sets of PCR primers. Each overlapping PCR fragment was sequenced using the fmol DNA sequencing system. RESULTS: tTG transcripts were detected in all samples. There was no difference in the coding sequence of tTG between the four control and eight coeliac patients, even though we observed differences in sequence between our study and the original published sequence. These differences have also been reported in sequences published subsequent to the original description. CONCLUSIONS: Polymorphisms in the tTG gene have not been observed in coeliac disease patients and therefore cannot explain the creation of neoepitopes.

Amino Acid Sequence↗

PubMed2000

Genetic screening in a large family with juvenile onset primary open angle glaucoma.

AIMS: A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members. METHODS: Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence. RESULTS: All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in the TIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family. CONCLUSION: The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.

Adolescent↗

PubMed2000

Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma.

BACKGROUND/AIMS: Inhibition of programmed cell death (apoptosis) is associated with increased tumour aggressiveness, and expression of Survivin, an antiapoptosis gene, in colorectal carcinomas may provide important prognostic information. PATIENTS/METHODS: Expression of Survivin messenger RNA was evaluated by reverse transcription-polymerase chain reaction in 144 colorectal carcinomas and 86 adjacent histologically normal mucosa samples from patients for whom long term follow up data were available. RESULTS: Survivin transcripts were detected in a significantly greater proportion of carcinomas (63.5%) than normal mucosa samples (29.1%; p<0.001). The prevalence of Survivin expression was independent of advancing pathological stage. Death due to recurrent cancer following curative resection was predicted independently by tumour expression of Survivin (hazard ratio (HR) 2.60; 95% confidence interval (95% CI) 1. 17-5.75) and lymph node metastases (HR 2.38; 95% CI 1.21-4.70). On stage wise analysis, the predictive value of Survivin expression was limited to patients with stage II colorectal carcinomas; those with Survivin negative tumours had a five year survival rate of 94.4% compared with 44.8% for patients with Survivin positive tumours (p=0. 004, log rank test). CONCLUSION: In patients with stage II colorectal carcinomas, Survivin expression provides prognostic information that may have important therapeutic implications.

Apoptosis↗

PubMed2000

Genomic organization of the human ubiquitin-conjugating enzyme gene, UBE2L6 on chromosome 11q12.

The human UBE2L6 gene encodes UbcH8(Kumar), a ubiquitin-conjugating enzyme (E2) highly simliar in primary structure to UbcH7 which is encoded by UBE2L3. Like UBC4 and UBC5 in yeast, these proteins demonstrate functional redundancy. Herein we report the intron/exon structure of UBE2L6. Comparison of the genomic organization of UBE2L6 with UBE2L3 demonstrates that these genes remain highly conserved at the genomic as well as at the protein level. We also describe the chromosomal localization of UBE2L6, which maps to chromosome 11q12.

Amino Acid Sequence↗

PubMed2000

Identification of a novel microsatellite marker tightly linked to the KAI-1 gene for predicting prostate cancer progression.

BACKGROUND: We have mapped the human prostate-specific membrane antigen (PSM) gene to the chromosome 11p11.2 region at 62.5 cM, a region which also contains the prostatic cancer metastasis suppressor gene KAI-1. The genetic marker D11S1344 has been utilised for loss of heterozygosity (LOH) studies on the KAI-1 gene in a large series of prostate cancer specimens. The results were negative and it was concluded that deletions of the KAI-1 gene were not involved in the development of the metastatic phenotype in these tumours. One possible explanation for this result could be that D11S1344 is not sufficiently tightly linked to the KAI-1 gene to detect small deletions. OBJECTIVE: To attempt to identify a genetic marker more tightly linked to the KAI-1 gene than D11S1344. METHODS: Yeast artificial chromosome (YAC) clones containing the KAI-1 gene and the neighbouring marker D11S1344 were analysed by the fluorescent in situ hybridisation technique. The human genomic inserts in these novel clones were sized by pulsed field gel electrophoresis. For more accurate mapping of the KAI-1 gene, YACs containing it were screened for polymorphic markers (including D11S1344) from the 11p11.2 region. RESULTS: The novel YAC clones localised exclusively to the 11p11.2 region, with single hybridisation signals compared to the dual signals consistently obtained with nearby PSM-containing YACs. All the KAI-1 clones found had small inserts (<300 kb). The only known microsatellite which gave amplification products with these YACs was D11S986 which has been mapped at 61.3 cM on human chromosome 11. CONCLUSIONS: We have precisely localised KAI-1 at 61.3 cM on human chromosome 11. This is some 1.2 cM away from the previously utilised LOH microsatellite marker, D11S1344. We suggest that the very tightly linked microsatellite D11S986 may be a more accurate marker to assess LOH of the KAI-1 gene and thus predict progression of prostate cancer. The region of genetic duplication around the PSM gene does not extend as far distally on 11p as KAI-1.

Antigens, CD↗

PubMed1999

The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1.

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.

Amino Acid Sequence↗

PubMed1999

Isolation and characterisation of a cDNA encoding the precursor for a novel member of the acyl-CoA dehydrogenase gene family.

A gene encoding the precursor for a novel member of the human acyl-CoA dehydrogenase (ACD) gene family has been isolated which maps to human chromosome 11q25. The cDNA contains an open reading frame of 1248 nucleotides encoding a predicted 415-amino-acid peptide, and shares considerable sequence similarity with other members of the ACD family.

Acyl-CoA Dehydrogenase↗

PubMed1999

Genotype/phenotype correlations for coagulation factor XIII: specific normal polymorphisms are associated with high or low factor XIII specific activity.

Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.

Abortion, Habitual↗

PubMed1999

Characterization of the mouse ubiquitin-conjugating enzyme gene UbcM4.

The ubiquitination pathway targets not only normal (short-lived) intracellular eukaryotic proteins for degradation when appropriate, but also serves to eliminate mutant/misfolded proteins from the cell. An understanding of the molecular basis of the interaction between the ubiquitin-conjugating enzymes (E2s), ubiquitin protein ligases (E3s), and target proteins is essential to explain the process in normal cellular function and in disease. UbcM4 is the mouse ortholog of the human E2, UbcH7, which can participate in the in vitro degradation of many proteins including p53. We describe the characterization of the mouse UbcM4 gene and the identification of a UbcM4 pseudogene. Four UbcM4 transcripts of approximately 0.7, 1.5, 2.1, and 2.6 kb, observed on Northern blots, are differentiated by their utilization of alternative UbcM4 polyadenylation sites. A single alternative splice variant cDNA, termed UbcM4Deltaex2, was also identified. The polypeptide encoded by UbcM4Deltaex2 is incapable of forming an ubiquitin-thioester in contrast to UbcM4, despite retaining the key cysteine residue essential for ubiquitin thioester formation and the active site consensus sequence that defines the ubiquitin-conjugating enzyme class. These observations are of particular relevance for analysis of UbcM4 function in vivo as our studies indicate that the targeted deletion of the coding exon absent in UbcM4Deltaex2 would produce an inactive UbcM4 protein and presents an alternative to disruption of its transcriptional initiation site/promoter region. Furthermore, it suggests that a similar strategy may be applicable to disrupt the function of other ubiquitin-conjugating enzymes in vivo.

Alternative Splicing↗