Naushin Waseem

Intracellular localization of the hepatitis B virus HBx protein

The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.

Haemophilia A mutations in the UK: results of screening one-third of the population.

One‐third of the UK haemophilia A population was screened to establish a national database of mutations and pedigrees and advance knowledge of the disease. The following mutations were found: 131 intron 22‐ and 13 intron1‐breaking inversions; 11 gross deletions and an insertion; 65 frameshifts; three in‐frame deletions and one insertion; 46 nonsense; 30 intronic mutations affecting splice sites and four generating new sites; 469 non‐synonymous mutations due to 203 different base substitutions of which four affected, and nine were predicted to affect, splicing; three promoter mutations; two synonymous exon 14 mutations possibly affecting splicing; two VWF mutations. Of the above mutations, 176 are not listed in the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Four gross deletions arose by non‐homologous end‐joining; we detected unexpected splicing in some mutations; substitution of amino acids conserved for less than 90 million years are rare; the risk of developing inhibitors for patients with nonsense mutations is greater when the stop codon is in the 3′ half of the mRNA; changes likely to generate splice sites causing frameshifts are over‐represented among non‐synonymous mutations associated with inhibitors; our data and those in HAMSTeRS enabled the size of the spectrum of specific mutations causing the disease to be estimated and to determine how much of it is known.

Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid‐phase fluorescent chemical cleavage of mismatch and examining additional non‐coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.

Isolation, sequence and expression of the gene encoding human keratin 13.

Keratins are a family of highly homologous proteins expressed as pairs of acidic and basic forms which make intermediate filaments in epithelial cells. Keratin 13 (K13) is the major acidic keratin, which together with K4, its basic partner, is expressed in the suprabasal layers of non-cornified stratified epithelia. The mechanism which allows mucosal-specific expression of this keratin remains unknown. To provide insight into the tissue-specific expression, we have isolated the human K13 gene by screening a chromosome 17 library with a specific K13 cRNA probe. Sequence analysis of unidirectional deletions produced by transposon Tn3 has revealed that the gene is 4601 nucleotides long and contains seven introns and eight exons. When driven by the CMV promoter, the gene produced K13 protein in MCF-7 cells, which normally do not express this protein. Two transcription-start sites were identified, the major being at 61 and the minor at 63 nucleotides upstream of ATG. The upstream sequence contained a TATA box and several other putative transcription factor binding sites. A single copy of the K13 gene was detected in the human genome by Southern hybridisation and polymerase chain reaction. K13 mRNA shows differential expression in cultured keratinocytes, and in A431 cells the RNA levels remained independent of calcium concentrations in the culture medium. Characterisation of the human K13 gene will facilitate elucidation of the molecular mechanism regulating K13 expression in mucosal tissues.

Identification of a novel keratin epitope: Evidence for association between non-helical sub-domains L12 during filament assembly

Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18) polypeptides. The assembly of these polypeptides into intermediate filaments is a complex multi-stage phenomenon that involves several levels of associations. These molecular associations are not very well characterized. Monoclonal antibodies (MAbs) with defined specificities can be used to probe these associations and to isolate various intermediates in the assembly pathway. Here we describe the specificity of a MAb LE65 that has been widely used in keratin expression studies. We report that the MAb LE65 does not recognize individual keratin polypeptides but it instead reacts with a complex of K8 with K18. The MAb also did not react with complexes of K8 or K18 with other keratins. By allowing the antibody to react with complexes reconstituted from keratin fragments plus the complementary keratin, we have mapped the MAb LE65 epitope on the L12 sub-domains of K18, residues 214-231, and K8, residues 234-265, which must associate together to achieve antibody binding. These results suggest that the non-helical linkers, L12, of complementary keratins associate directly during filament assembly. This would explain why microinjection of MAb LE65 has been shown to disrupt keratin filaments. Furthermore, it may also help to explain the mechanism of filament disruption in some skin blistering syndromes induced by spontaneous mutations in the L12 region.