Gene Function & Disease最新文献

Between one quarter and one third of all genes in eu- and prokaryotic organisms code for integral membrane proteins. Despite their eminent biological roles structural genomics projects so far excluded this class of proteins, mostly because of their amphiphilic character that imposes a variety of “non-standard” requirements for protein expression, purification, detergent-dependent solubilisation, and crystallization. Consequently, major obstacles for the structure determination of integral membrane proteins are the low success rates of 3D-crystallization and the number of membrane proteins which are available in amounts suitable for structural studies. While a variety of crystallization techniques for membrane proteins were developed during the last decade, no systematic approaches have yet been applied to augment the supply of new membrane proteins as candidates for 3D-crystallization. Several pilot projects applying structural genomics strategies on membrane proteins have now been initiated to close the increasing gap between genomic and structural information for this protein class.

The emerging ‘Structural Genomics’ initiatives provide novel opportunities to complement the rapidly increasing amount of genomic sequence data with the three-dimensional molecular structures of the coded genes. Many of the gene products exert their cellular functions combinatorially by interacting with multiple partners. Unravelling the molecular structures of these interactors provides the most useful information to investigate their involvement in cellular processes. To this end, the determination of structures exceeds the number of coded gene products by several orders of magnitude. ‘Structural Genomics’ offers opportunities to synergize European research in structural biology technologies, with a multitude of excellent centres currently available. In this contribution, current initiatives of the Hamburg Outstation of the European Molecular Biology Laboratory will be outlined.

Metalloproteins constitute a significant share of the proteome. Their structure determination and protein-protein interaction studies allow investigation of the role of metal cofactors and interpretation of the biophysical features and the biological function. The approach is that of genome browsing, expression of a few representative samples and partner proteins, structure determination (mainly by NMR, through quick protocols), and modeling of the other homologous proteins. The analysis of structures which in turn connect genes to protein function is presented for some proteins involved in copper homeostasis and for some classes of cytochromes c and ferredoxins.

Structural genomics (structural proteomics) initiatives aiming at the systematic, genome-directed structure analysis of proteins have recently been established in many locations worldwide. As a common international research endeavor, these initiatives thrive at the determination of protein structures representing all sequence families known in the biosphere. To reach this goal, a degree of international coordination of the structural genomics effort, as well as the development of high-throughput structure analysis methods and the provision of suitable research facilities are required. The Protein Structure Factory is a local, Berlin-based structural genomics project aiming at the structure determination of human proteins of presumed structural novelty and medical relevance. This project employs both NMR spectroscopy and X-ray crystallography for protein structure determination and aims at developing automated or semi-automated procedures for protein expression cloning and purification, and NMR spectral assignments. In addition, protein crystallography facilities are being set up at the third-generation synchrotron storage ring BESSY II in Berlin.

Genetic diseases can be caused by pure genetic factors or a combination of genetic and environmental factors. Mutations can occur in the autosomal chromosomes (chromosome 1−22), the sex chromosomes (X or Y), or the mitochondrial genome. Genetic diseases are transmitted from parents to offspring and can be categorized into two groups; monogenic and complex diseases. Monogenic diseases are caused by mutations in a single gene and complex diseases are caused by several genes in interaction with each other as well as with environmental factors. Systemic lupus erythematosus, SLE, is a systemic autoimmune disorder. The disease is characterized by chronic inflammation in different organ systems and autoantibodies against intracellular components such as dsDNA. The disease primarily affects women with a female to male ratio of 9:1. SLE is considered to be a complex disease, caused by interaction between genetic and environmental factors. In complex diseases multiple genetic factors with unknown mode of inheritance contribute to the disease. The susceptibility genes are thought to work together to cause the disease where neither gene is necessary nor sufficient. This review deals with the basis of the studies on genetics of complex diseases and what is known to date on the genetics of systemic lupus erythematosus.

Pseudoxanthoma elasticum (PXE) is an inherited systemic disorder of connective tissue, affecting the skin, the eyes, and the vascular system in a highly variable phenotypic expression. The PXE locus has been mapped to chromosome 16p13.1 and mutations in the ABCC6 gene (previously known as MRP6 or eMOAT), encoding a 1503 amino acids putative membrane transporter of unknown function, have recently been disclosed as the genetic defect responsible for PXE. We have identified a heterozygous missense mutation (G226R) in exon 7 of the ABCC6 gene in a PXE female patient, born from parents who were second cousins. Despite complete scanning of the gene, no further mutation was evident. A heterozygous profile was also found in the proband's unaffected children, the mutant peak being of much lower amplitude. However, haplotype homozygosity was confirmed at locus 16p13.1, using both, extragenic microsatellites (D16S3017 and D16S3060) and intragenic polymorphisms (V614A in exon 14 and R1268Q in exon 27) located 3′ from mutation G226R, in agreement with the known consanguinity in the family. Taken together, our data indicate that PCR products of exon 7 of the ABCC6 gene were amplified from more than two genomic copies. This further supports the existence of ABCC6 pseudogene(s) (ψABCC6) highly homologous to the 5′ end (exons 1−9) of the human ABCC6 gene. These results will prove invaluable when genotyping patients affected with PXE.

Gene regulation of fetal development is not well understood. In part, insulin and insulin-like growth factors (IGF) modulate placental steroid synthesis (PSS), which in turn modulates fetal growth. However, many of the genes that participate in this function remain to be identified. To find such genes, we examined the expression patterns of known IGF and placental steroid synthesis (IGF/PSS) genes in 1176 human cDNA libraries. We found a set of eight known IGF/PSS genes (PL-4, hCG, PAPP-A, EMBP, PLAP, P450 aromatase, P450scc, and 3-beta-HSD) that shared a highly similar expression profile across these libraries. We used these eight as bait in a search for other genes that showed very similar expression, and that might thus be related in function. We found ten genes closely co-expressed with the eight bait genes, but not previously reported as linked to IGF/PSS. Of these ten, six were previously reported as associated with cell growth in fetal and/or cancer tissues (malignant melanoma metastasis suppressor, PLAC-1, PSG10, PSG-beta1, serine palmitoyl transferase, and TONDU). Four are EST sequences, here named PLAC2, PLAC3, PLAC4, and PLAC5. Co-expression provides a method to identify which human genes are promising candidates for further experiments to determine their roles in fetal development.

Mutations in the gene of the gap junction protein Connexin 31 (CX31; other connexin genes abbreviated by CX+#, i.e. Connexin 30 = CX30) have been demonstrated to be responsible for both autosomal dominant and recessive nonsyndromic hereditary hearing impairment (NHHI). In this study, we assessed the prevalence of CX31 mutations in patients who had undergone cochlear implant surgery for profound sensorineural hearing loss and investigate the potential relationship between sequence alterations in CX31 and rehabilitative outcome. The single coding exon of CX31 was amplified by PCR from genomic DNA of cochlear implant patients. Of the 57 patients, 14 patients (25 %) had altered sequence in CX31; sequence analysis identified 15 single base changes in the 14 for a 13 % (15/114) incidence of variant allele frequency in the study population. Four distinct single nucleotide transitions were recognized including: one previously undocumented single nucleotide transition (250G → A) that resulted in an amino acid substitution at codon 84 (V84I) and three previously described single nucleotide polymorphisms (SNPs) (94C → T, 357C → T, and 798C → T). A single patient exhibited the 357C → T SNP in a homozygous state while the remaining patients' sequence variations were heterozygous. The novel V84I amino acid substitution occurred in the conserved second transmembrane domain of CX31 known to be critical for the regulation of voltage gating. However, the biologic consequence of this mutation and how it may relate to hearing loss is unknown. Rehabilitative outcome with cochlear implantation was similar in patients with and without CX31 mutations. Our data suggests that sequence alteration in CX31 is common in patients undergoing cochlear implantation and their rehabilitative outcome is unaffected.