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The very common GNB3 c.825CT polymorphism (rs5443) is present in approximately half of all human chromosomes. Significantly, the presence of the GNB3 825T allele has been strongly associated with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we, therefore, bioinformatically designed, using a Gene Tools(®) algorithm, a GNB3-specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ∼58% to ∼5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ∼42% to ∼95%). Our results demonstrate the potential use of a GNB3-specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties. Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum. During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6. Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation. Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S. pombe, C. elegans, and P. mariana. Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain. By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation.

The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper des-cribes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3′ RACE, a further 939 bp of cDNA sequence was obtained. The 1·9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism.

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

Background: The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the beta transducin subunit in the colour visual transduction process. A naturally occurring mutation 'D153del' in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. Principal Findings: Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the G beta 3 protein subunit that, together with different G gamma and G alpha subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. Significance: These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Molecular and morphological parameters of Serpula lacrymans isolates from various sites in the built environment in Europe and Australia were compared to similar parameters of ‘wild’ isolates from India, the Sumava Mountains (Czech Republic) and Mount Shasta (USA). The Indian, Czech Republic and all of the building isolates bar one showed identity in both molecular and morphological features. The Australian and the USA isolates (BF-050 and USA’94 respectively) showed specific morphological differences and could be separated on the basis of randomly amplified polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD PCR) with the USA isolate being least closely related to the S. lacrymans type strain of FPRL12C. ITS sequence data revealed two base differences between FPRL12C and BF-050 in the 673 sequenced, nine differences between FPRL12C and USA’94 and 16 differences between USA’94 and the closely related organism Serpula himantioides. The possible evolutionary relationships between the various isolates are discussed along with suggestions for the origin of S. lacrymans as a scourge of the built environment in many temperate areas of the world. > 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.

Restriction fragment length polymorphisms were investigated in five racial groups using the X chromosome probes DXS9 and DXS7. The allele frequencies of these polymorphisms showed significant differences and both DNA fragments were found to be highly polymorphic in the populations of south and southeast Asia. In the Marathi population of India, a rare allele B*3 (3 kilobases; kb) and an altered 7-kb fragment instead of the 6.6-kb constant band were found with DXS9. This is the first time that the rare B*3 allele is found in a non-European population.