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PubMed2005

Structural genomics of human proteins--target selection and generation of a public catalogue of expression clones.

BACKGROUND: The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells. RESULTS AND CONCLUSION: Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins.

Journal Article↗

PubMed2005

Structural genomics of thermotoga maritima proteins shows that contact order is a major determinant of protein thermostability.

Despite numerous studies, understanding the structural basis of protein stability in thermophilic organisms has remained elusive. One of the main reasons is the limited number of thermostable protein structures available for analysis, but also the difficulty in identifying relevant features to compare. Notably, an intuitive feeling of "compactness" of thermostable proteins has eluded quantification. With the unprecedented opportunity to assemble a data set for comparative analyses due to the recent advances in structural genomics, we can now revisit this issue and focus on experimentally determined structures of proteins from the hyperthermophilic bacterium Thermotoga maritima. We find that 73% of T. maritima proteins have higher contact order than their mesophilic homologs. Thus, contact order, a structural feature that was originally introduced to explain differences in folding rates of different protein families, is a significant parameter that can now be correlated with thermostability.

Amino Acid Sequence↗

PubMed2003

Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.

As the field of structural genomics continues to grow and new technologies are developed, novel strategies are needed to efficiently crystallize large numbers of protein targets, thus increasing output, not just throughput [Chayen & Saridakis (2002). Acta Cryst. D58, 921-927]. One strategy, developed for the high-throughput structure determination of the Thermotoga maritima proteome, is to quickly determine which proteins have a propensity for crystal formation followed by focused SeMet-incorporated protein crystallization attempts. This experimental effort has resulted in over 320 000 individual crystallization experiments. As such, it has provided one of the most extensive systematic data sets of commonly used crystallization conditions against a wide range of proteins to date. Analysis of this data shows that many of the original screening conditions are redundant, as all of the T. maritima proteins that crystallize readily could be identified using just 23% of the original conditions. It also shows that proteins that contain selenomethionine and are more extensively purified often crystallize in distinctly different conditions from those of their native less pure counterparts. Most importantly, it shows that the two-tiered strategy employed here is extremely successful for predicting which proteins will readily crystallize, as greater than 99% of the proteins identified as having a propensity to crystallize under non-optimal native conditions did so again as selenomethionine derivatives during the focused crystallization trials. This crystallization strategy can be adopted for both large-scale genomics programs and individual protein studies with multiple constructs and has the potential to significantly accelerate future crystallographic efforts.

Bacteria↗

PubMed1988

Two-dimensional DNA gel electrophoresis as a method for analysis of eukaryotic genome structure: evaluation using Tetrahymena thermophila DNA.

There is growing interest in mapping and analyzing complete eukaryotic genomes. Yee and Inouye (in Experimental Manipulation of Gene Expression, pp. 279-290, Academic Press, New York) demonstrated that bacterial chromosomes can be resolved into interpretable patterns of DNA fragments by means of restriction enzyme digestion and electrophoresis in two dimensions. We have begun to explore applications of this procedure to analysis of eukaryotic genomes, which are far more complex. Tetrahymena thermophila was selected as a model organism because its genome is small, roughly equivalent to that of a single human chromosome. In addition, each Tetrahymena cell contains two nuclei which differ in sequence composition and methylation. Our results demonstrate that the Tetrahymena genome can be resolved into complex patterns of fragments in two dimensions. Hybridization to Southern blots of these gels with a multiply repeated sequence probe yielded analyzable patterns of a subset of the genome. The blots reveal alterations in genome structure due to methylation and rearrangement. Future extensions of the method are discussed.

Animals↗

PubMed2026

Structural genomics sheds light on protein functions and remote homologs across the insect tree of life.

Protein structure bridges the sequence-function relationship, enabling deep exploration of biological processes across diverse organisms. Insects, the most diverse animal lineage, accounting for over 50% of all described animal species, provide an exceptional system for exploring sequence-structure-function relationships. Here, we reconstructed a comprehensive and well-resolved phylogeny of 4854 insects, spanning all orders. Leveraging this framework, we created an atlas of 13.29 million predicted protein structures from 824 representative species, including 11.63 million newly predicted structures. Structural clustering revealed that proteins with divergent sequences but similar structures could be effectively grouped together. Structural similarity searches against proteins with well-characterized functions yielded annotations for 7.61 million insect proteins, including up to 14% of previously unannotated proteins. We further identified 750 million remote homologs between insect proteins, many of which trace back to ancient branches of the insect phylogeny. Remarkably, despite extensive sequence divergence, cGAS-like receptors (cGLRs) were structurally conserved across all 824 insects. Experimental assays demonstrated that these structurally identified cGLRs play a crucial role in antiviral defense in the yellow fever mosquito. Our findings highlight the significance of structural genomics for understanding protein function and evolution across the tree of life.

Animals↗

PubMedDate not supplied

[Biological properties and genome structure of recombinant strains of influenza A virus differing in their degree of human virulence].

The genome structure and some biological properties of parental and recombinant strains of influenza viruses differing by degrees of virulence for man were studied. The vaccine A/Leningrad/9/46 (H0N1) strain was shown to be markedly toxic and pathogenic for mice in contrast to influenza A/Brazil/11/78 and A/Victoria/35/72/50 viruses completely devoid of these properties. The recombinants deriving all 6 genes of the inner proteins of influenza A/Leningrad/9/46 strains and hemagglutinin and neuraminidase of the epidemic A/Brazil/11/78 strain have completely lost their pathogenicity for mice but acquired marked virulence for man. No such effect was observed in recombination of A/Brazil/11/78 with another vaccine strain, A/Victoria/35/72/50. It is suggested that the toxic properties of influenza virus are coded for in genes of internal proteins whereas their manifestation in one or another host species depends on the function of surface polypeptides.

Animals↗

PubMed1997

Differential changes in genome structure and expression of the mdr gene family in multidrug-resistant murine erythroleukemia cell lines.

The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

The genome structure of kyuri green mottle mosaic tobamovirus and its comparison with that of cucumber green mottle mosaic tobamovirus.

The genome of the Y strain of kyuri green mottle mosaic virus (KGMMV-Y) has been completely sequenced. Its genomic structure and sequence show it to be a typical tobamovirus, that is closest to, but distinct from, that of cucumber green mottle mosaic tobamovirus (CGMMV). The genomic sequence of KGMMV-Y was compared in detail with that of the SH strain of CGMMV. The sequences of their 5'- and 3'-untranslated regions were 74% and 63% identical. The amino acid sequences of the shorter and longer (read through) RNA replicase components, movement protein (MP) and coat protein (CP) were 58, 58. 60 and 46% identical, respectively. The KGMMV-Y genome sequence was also compared partly to that of another strain of KGMMV, KGMMV-C. The CP sequences of KGMMV-Y and KGMMV-C differed by 20 amino acid residues, suggesting that their relationship is more distant than the relationship between CGMMV-SH and CGMMV-W whose CP sequences are identical. The MPs of KGMMV-Y and KGMMV-C, however, differ only by one amino acid residue, although three amino acid substitutions are present in the MPs between CGMMV-SH and CGMMV-W. Two long stretches, one in the RNA replicase and the other in the MP, were highly conserved in KGMMV and CGMMV.

Amino Acid Sequence↗

PubMed2000

Biological function made crystal clear - annotation of hypothetical proteins via structural genomics.

Many of the gene products of completely sequenced organisms are 'hypothetical' - they cannot be related to any previously characterized proteins - and so are of completely unknown function. Structural studies provide one means of obtaining functional information in these cases. A 'structural genomics' project has been initiated aimed at determining the structures of 50 hypothetical proteins from Haemophilus influenzae to gain an understanding of their function. Each stage of the project - target selection, protein production, crystallization, structure determination, and structure analysis - makes use of recent advances to streamline procedures. Early results from this and similar projects are encouraging in that some level of functional understanding can be deduced from experimentally solved structures.

Bacterial Proteins↗

PubMed2004

cDNA cloning, genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig.

A novel cDNA has been isolated from pig parotid glands by 3' and 5' rapid amplification of cDNA ends and designated parotid secretory protein (PSP). The open reading frame of this cDNA covers 714 bases, encoding 238 amino acids, which show 56% identity with human PSP at the level of the primary protein structure. The PSP genomic sequence comprises eight exons and seven introns, is approximately 22 kb in size, determined by sequencing, and maps to pig chromosome 17q21-q23. RT-PCR, dot blot, and Northern blot analyses demonstrated that PSP is strongly expressed in parotid glands, but is not present in heart, liver, lung, kidney, muscle, or stomach. A search for functionally significant protein motifs revealed consensus sequences for casein kinase II phosphorylation and N-myristoylation. We observed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is similar to the leucine zipper.

Amino Acid Sequence↗

PubMed2002

Genomic structure and eight novel exonic polymorphisms of the human N-cadherin gene.

Analysis of the detailed genomic structure of human N-cadherin revealed that the 16-exon gene is more than 72 kb in length and that it consists of a mosaic of exons. Five repeated cadherin domains, a transmembrane domain, and a cytoplasmic domain are encoded by exons 4 to 13, 13 and 14, and 14 to 16, respectively. A search for molecular variants in the entire coding region in 96 Japanese individuals resulted in the identification of eight sequence polymorphisms including three CCT- or GCC-type trinucleotide repeat polymorphisms adjacent to the initiation codon and five other novel single-nucleoticle polymorphisms (SNPs) in the coding region. Three of the five SNPs accompanied an amino acid substitution: Ala118Thr, Ala826Thr, and Asn845Ser. Knowlege of the fine gene structure and eight novel polymorphisms will be useful for the genetic study of the role of N-cadherin in diseases involving cell adhesion in the brain and in cardiomyocytes.

Brain Neoplasms↗

PubMed2002

Complete genomic structure of human rpS3: identification of functional U15b snoRNA in the fifth intron.

Analysis of the complete genomic structure of the human ribosomal protein S3 (rpS3) gene revealed the presence of a functional U15b snoRNA gene in its intron. Human ribosomal protein S3 (rpS3) gene of 6115 bp long has been identified to contain six introns and seven exons in this study. The first and fifth introns of human S3 gene contain functional U15 snoRNA genes. Although Xenopus and Fugu counterparts also have six introns and seven exons, S3 gene of Fugu contains two functional U15 snoRNAs in the fourth and sixth introns and two pseudo genes for U15 snoRNAs in the first and fifth introns. In Xenopus S1 gene encoding ribosomal protein S3, however, three of its six introns contain U15 snoRNA gene sequence. Sequence comparison of the U15 genes from Xenopus, Fugu and human revealed that the regions involved in binding to 28S rRNA and the consensus sequence (C, D and D' boxes) for snoRNAs are highly conserved among those genes from these three species. Human U15a and U15b RNAs which are derived from the first and the fifth introns, respectively, have been identified to be functional by microinjection of human U15a and U15b snoRNAs into Xenopus oocyte. Northern blot and primer extension analyses confirm that human U15b snoRNA is expressed in vivo.

Animals↗

PubMed2003

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes, Tact1 and Tact2.

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.

Actins↗

PubMed1999

Transforming growth factor-alpha (TGFA): genomic structure, boundary sequences, and mutation analysis in nonsyndromic cleft lip/palate and cleft palate only.

Transforming growth factor-alpha (TGFA) has been proposed as a candidate gene in the etiology of nonsyndromic cleft lip with or without cleft palate (NS-CL/P) and of nonsyndromic cleft palate only (NS-CPO). Biologic support for a role of TGFA arises from its presence at high levels in the epithelial tissue of the medial edge of the palatal shelves at the time of shelf fusion in mice. Genetic support for the role of TGFA in clefting comes from the reported association of TGFA alleles with human NS-CPO and NS-CL/P. In this study we report the sequence and structure of human genomic TGFA and the search for causal TGFA mutations in 250 individuals with NS-CL/P or NS-CPO by conformational analysis of the coding sequence, splice junctions, and a portion of the 3' untranslated region strongly homologous between human and mouse. We confirm that human TGFA is composed of six exons and here report several new sequence substitutions and their frequencies. Five variants in conserved segments may represent rare causes for clefting in humans and provide support for the role of TGFA in facial morphogenesis.

Animals↗

PubMed2001

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures.

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.

Base Sequence↗

PubMed2006

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices.

BACKGROUND: The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. RESULTS: In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. CONCLUSION: On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic changes, such as exonic indel mutations and gene duplications are less rare than previously thought and that these also occur within fungal populations.

Base Sequence↗

PubMed1997

Elastin: genomic structure and point mutations in patients with supravalvular aortic stenosis.

We describe the complete exon-intron structure of the human elastin (ELN) gene located at chromosome 7q11.23. There are 34 exons occupying approximately 47 kb of genomic DNA. All exons are in-frame, allowing exon skipping without disrupting the reading frame. Microsatellites are located in introns 17 and 18. Deletions of all or large parts of the ELN gene have been previously reported in two patients with supravalvular aortic stenosis (SVAS), and SVAS is also a frequent feature of Williams syndrome, where patients are hemizygous for ELN. We list primer pairs for amplifying each exon, with flanking intron, from genomic DNA to allow detection of point mutations in the ELN gene. We show that some patients with isolated SVAS have point mutations that are predicted to lead to premature chain termination. Knowledge of the genomic structure will allow more extensive mutation screening in genomic DNA of patients with SVAS and other conditions.

Amino Acid Sequence↗

PubMed2001

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics.

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.

Circular Dichroism↗