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PubMed2004

[Characterization of genomic structure and mutation analysis of SMARCA1 gene in a Smith-Fineman-Myers syndrome family].

The study is to determine the genomic structure and the role of SMARCA1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member1, SMARCA1) in the etiology of Smith-Fineman-Myers syndrome (SFMS). By comparing the cDNA sequence of SMARCA1 with the genomic sequences, genomic structure of SMARCA1 was determined, and conformed by amplifying and sequencing the sequences of exons and splicing junction. The results show that the genomic sequence of SMARCA1 gene exceeds 71.7 kb in length, and contains 24 exons and 23 introns. All the exon/intron boundaries follow the GT-AG rule and are in good agreement with the exon/intron consensus sequence. The characterization of genomic structure of SMARCA1 gene allows us to detect disease-causing mutation within the gene and further study its biological function. The open reading frame of SMARCA1 was detected for mutation by PCR amplification and direct sequencing in affected males from SFMS family in Shandong China. The disease in SFMS family from Shandong is not caused by the mutation within open reading frame of SMARCA1 gene.

Abnormalities, Multiple↗
PubMed2001

Characterization of the genomic structure of the human neuronal nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster and identification of novel intragenic polymorphisms.

Genes coding for the alpha5, alpha3, and beta4 subunits (CHRNA5, CHRNA3, and CHRNB4) of the neuronal nicotinic acetylcholine receptors (nAChRs) are clustered on chromosome 15q24. Linkage of this chromosomal region to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), an idiopathic partial epilepsy, was reported in one family. Moreover, mutations in other neuronal nAChR subunit genes coding for the alpha4 (CHRNA4) and the beta2 (CHRNB2) subunits were associated with ADNFLE. Apart from the exon-intron structure of CHRNA3, the genomic organization of this gene cluster was unknown, making comprehensive mutational analyses impossible. The genomic structure of CHRNA5 and CHRNB4 is here reported. Moreover, two hitherto unknown introns were identified within the 3' untranslated region of CHRNA3, causing a partial tail-to-tail overlap with CHRNA5. Four novel intragenic polymorphisms were identified and characterized in the cluster.

Amino Acid Sequence↗
PubMed2005

Structural genomics of minimal organisms and protein fold space.

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.

Bacterial Proteins↗
PubMed2001

Structural genomics: opportunities and challenges.

Following the complete genome sequencing of an increasing number of organisms, structural biology is engaging in a systematic approach of high-throughput structure determination called structural genomics to create a complete inventory of protein folds/structures that will help predict functions for all proteins. First results show that structural genomics will be highly effective in finding functional annotations for proteins of unknown function.

Crystallography, X-Ray↗
PubMed2005

Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima.

Studies of the structural basis of protein thermostability have produced a confusing picture. Small sets of proteins have been analyzed from a variety of thermophilic species, suggesting different structural features as responsible for protein thermostability. Taking advantage of the recent advances in structural genomics, we have compiled a relatively large protein structure dataset, which was constructed very carefully and selectively; that is, the dataset contains only experimentally determined structures of proteins from one specific organism, the hyperthermophilic bacterium Thermotoga maritima, and those of close homologs from mesophilic bacteria. In contrast to the conclusions of previous studies, our analyses show that oligomerization order, hydrogen bonds, and secondary structure play minor roles in adaptation to hyperthermophily in bacteria. On the other hand, the data exhibit very significant increases in the density of salt-bridges and in compactness for proteins from T.maritima. The latter effect can be measured by contact order or solvent accessibility, and network analysis shows a specific increase in highly connected residues in this thermophile. These features account for changes in 96% of the protein pairs studied. Our results provide a clear picture of protein thermostability in one species, and a framework for future studies of thermal adaptation.

Bacterial Proteins↗
PubMed1989

Genome structure of cottontail rabbit herpesvirus.

The genome structure of a herpesvirus isolated from primary cultures of kidney cells from the cottontail rabbit Sylvilagus floridanus was elucidated by using electron microscopy and restriction enzyme analysis. The genome, which was about 150 kilobase pairs long and which had an average G + C composition of 45%, consisted of two regions with unique base sequences (54 and 47 kilobase pairs) enclosed by reiterations of a 925-base-pair sequence with a variable copy number. The internal repeats were in opposite polarity with respect to the terminal repeats, and both unique regions underwent inversion. The nucleotide sequence of the repeat unit was determined, and virion DNA termini were precisely localized within this sequence. Elements showing homology with the cleavage-packaging signals common to other herpesviruses were detected. The data indicate that this virus is different from the previously described herpesvirus sylvilagus.

Animals↗
PubMedDate not supplied

Target selection and determination of function in structural genomics.

The first crucial step in any structural genomics project is the selection and prioritization of target proteins for structure determination. There may be a number of selection criteria to be satisfied, including that the proteins have novel folds, that they be representatives of large families for which no structure is known, and so on. The better the selection at this stage, the greater is the value of the structures obtained at the end of the experimental process. This value can be further enhanced once the protein structures have been solved if the functions of the given proteins can also be determined. Here we describe the methods used at either end of the experimental process: firstly, sensitive sequence comparison techniques for selecting a high-quality list of target proteins, and secondly the various computational methods that can be applied to the eventual 3D structures to determine the most likely biochemical function of the proteins in question.

Amino Acid Motifs↗
PubMed2005

Characterisation of the genomic structure of chick Fgf8.

The Fgf8 gene encodes a series of secreted signalling molecules important in the normal development of the face, brain and limbs. The genomic structure of the chick Fgf8 gene has been analysed and compared to the human and mouse sequences. Divergence between the chick, human and mouse genomic structure was observed. Data indicates that the long alternatively spliced form of exon 1b observed in mouse and exon 1c observed in human and mouse do not exist in the chick Fgf8 gene. RT-PCR analysis indicates that chick Fgf8, like its mouse and human counterpart is alternatively spliced. This data along with the genomic structure data indicates that in the chick there are only two isoforms of Fgf8. This is in contrast to the human and mouse, where evidence suggests that there are 4 and 8 isoforms, respectively. Approximately 400 bp of intron 1d is highly conserved between chick, human and mouse genomic sequences. Using TRANSFAC possible conserved regulatory element binding sites within this domain were identified.

Alternative Splicing↗
PubMed2003

NMR and structural genomics.

The role of NMR in structural genomics is outlined, with particular emphasis on using protein domains as targets. Strategies for domain expression, characterization, and labeling are presented.

Genomics↗
PubMed2004

Structural genomics on membrane proteins: the MePNet approach.

Of the 25,000 crystal structure entries deposited in public databases less than 1% are for membrane proteins; this gives a clear indication of the difficulties relating to structural biology and determination of this type of protein. To date, structural genomics networks have mainly focused on soluble proteins, despite the fact that membrane proteins constitute 60 to 70% of current drug targets. Technological improvement on a broad basis is the key to higher success rates, but this is generally not possible unless large functional networks are established. Several structural genomics initiatives, concentrating partly or fully on membrane proteins, have been established during the last few years and the Membrane Protein Network (MePNet; BioXtal, Switzerland) focuses specifically on the pharmaceutically attractive G protein-coupled receptors (GPCRs). Within MePNet, over 100 full-length GPCRs have been subjected to overexpression in bacterial, yeast and mammalian cells, followed by purification and crystallization studies.

Animals↗
PubMed2002

The TB structural genomics consortium crystallization facility: towards automation from protein to electron density.

The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored p50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake&wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.

Automation↗
PubMed2004

Refolding strategies from inclusion bodies in a structural genomics project.

The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.

Genome, Fungal↗
PubMed2002

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings.

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (<five minutes of computational time) and are in complete agreement with corresponding NOE-based assignments. Orientational constraints derived from RDCs are used simultaneously to assemble sub-structures into an RNA tertiary conformation. Through enhanced speeds of application and reduced reliance on chemical shift dispersion, this RDC-based approach lays the foundation for rapidly determining RNA conformations in a structural genomics context, and may increase the size limit of RNAs that can be examined by NMR.

Base Sequence↗
PubMed1993

Analysis of MHC genomic structure and gene content between HLA-B and TNF using yeast artificial chromosomes.

To characterize the genomic structure and gene content between HLA-B and TNF we studied three overlapping yeast artificial chromosome (YAC) clones derived from the Boleth cell line carrying the 62.1 MHC ancestral haplotype (AH). In particular, we have evaluated the stability of haplospecific sequence motifs (haplospecific geometric elements) in YACs. By PCR and gene scanning we show that the two larger YAC clones (318G11 and 8F2) exhibit features characteristic of the 62.1 AH carried by the parental cell line as well as those of a separate cell line (BSM) which also carries 62.1. It is concluded that a highly polymorphic duplicated region is represented faithfully in spite of the fact that it contains sequences that could be interpreted as simple repeats liable to mutations. In contrast, microsatellites AFM031 and AFM158, which are also located in the MHC, showed greater instability and the structure in the YACs was not consistent with genomic structure. Likewise, different examples of the same ancestral haplotype show differences at these microsatellite loci. We conclude that polymorphic repeat sequences may exhibit a range of stabilities in vivo and that this may be reflected in their stability in YACs. Northern blotting using the largest YAC clone revealed several novel transcripts which await localization.

Blotting, Northern↗
PubMed2006

The rate, not the spectrum, of base pair substitutions changes at a GC-content transition in the human NF1 gene region: implications for the evolution of the mammalian genome structure.

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.

Animals↗
PubMed2006

RIKEN structural genomics beamlines at the SPring-8; high throughput protein crystallography with automated beamline operation.

RIKEN Structural Genomics Beamlines, BL26B1 & BL26B2 at the SPring-8, have been constructed for the structural genomics research. The main feature of the beamline is full automation of the successive data collections to maximize the beam-time efficiency. The beamline optics adopted a standard design commonly used for the SPring-8 bending magnet beamlines. Beamline instruments are operated by centralized control system through the computer network to achieve the automatic operation. The core part of the beamline development is a sample management system composed of sample changer robots for laboratory and beamline, and a networked sample database. BL26B1 has started user operation, and the automatic operation with the sample management system has been implemented at BL26B2.

Crystallography, X-Ray↗