Gene Function & Disease最新文献

Holoprosencephaly (HPE) is the most common structural congenital forebrain malformation in humans and is associated with mental retardation and craniofacial abnormalities. In HPE, the cerebral hemispheres fail to separate into distinct right and left halves. The condition is etiologically heterogeneous, as multiple genes and environmental factors are associated with the condition. Autosomal dominant HPE can be caused by mutations in Sonic Hedgehog (SHH). Smoothened (SMOH; human gene) encodes a transmembrane protein that acts in SHH signal transduction. Because of the critical role of SMOH in the SHH pathway, we performed mutation analysis of this gene in familial and sporadic cases of HPE. Although we identified a number of nucleotide changes in SMOH in affected patients, none of these changes is likely to be pathogenic. Thus, haploinsufficiency for SMOH is unlikely to be a significant cause of human HPE in live-born infants.

The adenosine A_2B receptor is found on human lung mast cells and is believed to mediate the bronchoconstriction in response to adenosine characteristic of asthmatics. As such it represents an attractive therapeutic target for asthma and allergic rhinitis. As genetic variability in drug targets may affect an individual's response to treatment, the adenosine A_2B receptor gene was analyzed for polymorphisms and their prevalence defined in a Caucasian population. The coding region of the A_2B receptor gene as well as the intron-exon boundaries of the gene were found to be free of genetic variation. Three single nucleotide polymorphisms were identified in the promoter region of the gene, one of which created a BamHI restriction fragment length polymorphism. The prevalence of the three single nucleotide polymorphisms in an unselected population was <5%. The prevalence of the BamHI polymorphism was investigated in a British population of 40 asthmatics and 40 non-asthmatics: no excess of the BamHI variant was observed in asthmatic subjects. The data indicates that any variability in drug response would likely not be due to direct structural variation of the A_2B receptor protein. Further, polymorphism at the A_2B receptor genomic locus is unlikely to contribute to the population risk of developing asthma.

Cell adhesion and matrix remodeling are elements in many diseases, ranging from atherosclerosis and fibrosis to metastatic cancer. However, many genes that participate in these processes have not yet been identified. To find such genes, we looked for previously uncharacterized genes that are co-expressed with known cell adhesion and matrix remodeling genes. The known genes in this study included MMP2, TIMP3, BM-40, chondroitin, connective tissue growth factor, fibromodulin, IGFBP5, laminin, MGP, myosin light chain kinase, several collagens, and other matrix and adhesion proteins. We found eight previously uncharacterized genes, here named MXRA1 through MXRA8, that were strongly co-expressed with these known adhesion and matrix genes. Five of the MXRA genes have a significant similarity to uncharacterized cDNA sequences or predicted proteins listed in the Genbank database, but otherwise show distant or no sequence similarity to genes with known function. Subsequent to our entry of the MXRA gene sequences in the Genbank, three of the eight genes have been independently described by other researchers: MXRA2 is α-parvin, a cell-matrix adhesion protein, MXRA4 is a C1 complement component receptor involved in cell adhesion, and MXRA5 is adlican, an adhesion proteoglycan. The analysis described here provides further evidence for the role of these genes in adhesion and matrix remodeling.

We stimulated differentiation of purified rat neural crest stem cells (NCSCs) into smooth muscle cells (SMCs) in culture, then subtracted NCSC sequences from SMC sequences to make a cDNA library specific for differentiating smooth muscle cells. Sequence analysis of the library shows that a large subset of clones is strongly associated with smooth muscle biology, confirming the overall success of the differentiation and subtraction procedures. Of this subset of clones, more than half encode proteins that have previously been shown to be upregulated in atherosclerotic or injured vascular smooth muscle as compared to normal vascular smooth muscle. Thus, a set of genes activated in differentiating smooth muscle of the neural crest lineage is also activated in atherosclerotic or injured vascular smooth muscle.

Organ biopsies from kidney, liver, intestine or heart are presently only evaluated for pathologic lesions using formalin-fixation and paraffin-embedding. Changes in pattern and levels of gene expression underlying and preceding these lesions have been disregarded, since a quantitative assay system using minute paraffinized tissue samples with minimal amounts of RNA is currently unavailable. This paper describes such an assay using formalin-fixed, paraffinized biopsies from human glomeruli after laser-microdissection. Choosing proliferative glomerular lesions expected to express platelet-derived growth factor-β receptor (PDGF-βR), we investigated whether vascular cell adhesion molecule-1 (VCAM-1), a marker of the inflammatory signal pathway, was coexpressed, indicating the potential to progress an already existing lesion. RNA extraction procedures including DNAse pretreatment were pretested on microdissected mouse glomeruli. β-actin, not upregulated in cell culture by TNF (tumor necrosis factor) or PDGF stimulation, served as housekeeping gene. By qualitative real-time (RT)-PCR, transcription of both genes was detectable using only one microdissected glomerular section. Quantitative PCR for VCAM-1 revealed unsuspected increased levels in the same cases positive by qualitative RT-PCR, not detectable by morphologic analysis or immunohistochemistry. Thus, quantitative gene expression analysis is possible in paraffinized biopsies from minute tissue samples and will provide such important information as unexpected gene expression causing a potential progression of an existing lesions.

Zinc finger transcription factors play essential roles in neural crest cell development. Even subtle disruptions of the function of these genes could contribute significantly to complex developmental phenotypes such as the multigenic disorder Hirschsprung disease (HSCR), a congenital failure of enteric neurogenesis. Although germline mutations in the RET proto-oncogene are the most common cause of familial disease, at least 8 genes including transcription factors have been implicated in sporadic HSCR to date. Thus, a further group of candidate genes are those involved in regulating expression of known HSCR genes. In this study, we have characterized the ZNF358 zinc finger gene, a human orthologue of the mouse gene zfend, which is expressed in early developmental stages in the gut and in the neural folds at the time of neural crest differentiation. ZNF358 represents a putative transcription factor with DNA binding activity confered by 9 Cys₂His₂ zinc fingers. Although we did not detect disease associated mutations in a panel of HSCR patients, we did identify a novel variable sequence within the coding region of ZNF358 that would result in a deletion of nine amino acids from a polyalanine domain. Our molecular model suggests that this variant could alter protein tertiary structure and the ability of ZNF358 to regulate transcription. Together, our data suggest that, while ZNF358 may be involved in the regulation of genes such as those necessary for development or differentiation of neural crest cells, it does not play an obvious role in HSCR.

Determining the structures of proteins of entire proteomes is a formidable endeavour. High throughput structural genomics correspondingly requires the development of highly efficient methods to streamline every step in progressing from the individual gene expression to refined three-dimensional protein structure. A major bottleneck in structural biology is the production of sufficient amounts of correctly folded and functional protein. Here, we present a simple “Plug and Play”-expression system that allows for parallel gene expression in E. coli, yeast and insect cells to rapidly select the best strategy for the production of high-quality protein.

The TRANSFAC system of databases provides information about the molecular mechanisms of transcriptional regulation and pathological dysregulation, certain aspects of chromatin structure, and signal transduction. Integrating this information into a comprehensive system allows to model complex regulatory networks of experimentally known as well as hypothetical components, the latter ones being predicted by the application of state-of-the-art bioinformatics tools. Altogether, it will provide core information with which it ill be possible to tackle most of the problems in the field of “functional genomics”.