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PubMed2000

Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2).

Spinal muscular atrophy (SMA), a common motor neuron disease in humans, results from loss of functional survival motor neuron (SMN1) alleles. A nearly identical copy of the gene, SMN2, fails to provide protection from SMA because of a single translationally silent nucleotide difference in exon 7. This likely disrupts an exonic splicing enhancer and causes exon 7 skipping, leading to abundant production of a shorter isoform, SMN2Delta7. The truncated transcript encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. This report describes the identification of an in vivo regulator of SMN mRNA processing. Htra2-beta1, an SR-like splicing factor and ortholog of Drosophila melanogaster transformer-2, promoted the inclusion of SMN exon 7, which would stimulate full-length SMN2 expression. Htra2-beta1 specifically functioned through and bound an AG-rich exonic splicing enhancer in SMN exon 7. This effect is not species-specific as expression of Htra2-beta1 in human or mouse cells carrying an SMN2 minigene dramatically increased production of full-length SMN2. This demonstrates that SMN2 mRNA processing can be modulated in vivo. Because all SMA patients retain at least one SMN2 copy, these results show that an in vivo modulation of SMN RNA processing could serve as a therapeutic strategy to prevent SMA.

Animals↗
PubMed2006

Turning junk into gold: domestication of transposable elements and the creation of new genes in eukaryotes.

Autonomous transposable elements, generally considered as junk and selfish, encode transposition proteins that can bind, copy, break, join or degrade nucleic acids as well as process or interact with other proteins. Such a repertoire of activities might be of interest for the host cell. There is indeed substantial evidence that mobile DNA can serve as a dynamic reservoir for new cellular functions. Transposable element genes encoding transposase, integrase, reverse transcriptase as well as structural and envelope proteins have been repeatedly recruited by their host during evolution in most eukaryotic lineages. Such domesticated sequences protect us against infections, are necessary for our reproduction, allow the replication of our chromosomes and control cell proliferation and death; others are essential for plant development. Many new candidates for domesticated sequences have been revealed by sequencing projects. Their functional analysis will uncover new aspects of evolutionary alchemy, the turning of junk into gold within genomes.

Animals↗
PubMed2006

Comparative genomic hybridization reveals genetic progression of oral squamous cell carcinoma from dysplasia via two different tumourigenic pathways.

To clarify the genetic pathway(s) involved in the development and progression of oral squamous cell carcinoma (OSCC), as well as the relationship between genetic aberrations and biological characteristics of OSCC tumours, comparative genomic hybridization was used to analyse genetic alterations in both primary OSCCs and adjacent dysplastic lesions of the same biopsy specimens from 35 patients. Gain of 8q22-23 was the most frequent alteration in both OSCC and mild dysplasia, and was considered the earliest event in the process of oral tumourigenesis. The average number of DNA sequence copy number aberrations (DSCNAs) increased with progression from mild dysplasia to invasive carcinoma (r = 0.737, n = 70, p < 0.001). OSCC samples were classified as having a large or small number of DSCNAs (OSCC-L, 21.4 +/- 4.7 DSCNAs or OSCC-S, 10.0 +/- 1.7 DSCNAs, respectively; p < 0.0001). Gains of 3q26-qter, 8q, 11q13, 14q, and 20q and losses of 4q, 5q12-22, 6q, 8p, 13q, and 18q22-qter were common to OSCC-L and OSCC-S. Gains of 5p15, 7p, 17q11-22, and 18p and losses of 3p14-21, 4p, and 9p were detected exclusively in OSCC-L. The average number of DSCNAs depended on whether the samples showed OSCC- L or dysplasia plus OSCC-L, or showed OSCC-S or dysplasia plus OSCC-S (p = 0.001). Gain of 5p15 and losses of 4p and 9p were detected even in dysplastic lesions adjacent to OSCC-L samples. Loss of 4p was associated with node metastasis by multivariate analysis (p = 0.013). OSCC-L tumours were more often T3-T4 stage tumours than T1-T2 stage tumours (p = 0.03). These findings suggest that two different types of OSCC, OSCC-L associated with high-stage cancer and OSCC-S associated with low-stage cancer, arise from different types of dysplasia via different genetic pathways.

Aged↗
PubMed1979

The regulation of RNA synthesis in yeast IV. Synthesis of double-stranded RNA.

A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.

Cell Division↗
PubMed2004

Gene conversion and functional divergence in the beta-globin gene family.

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication-degeneration-complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d(N)/ d(S) (omega) rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the beta-globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate beta-globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohta's model and the DDC model by examining selective pressures along lineages in a phylogeny of beta-globin genes in eutherian mammals. We did not find significant evidence for an increase in the omega ratio following major duplication events in this family. However, one exception to this pattern was the duplication of gamma-globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of beta-globin genes evolved under a nonepisodic process of functional divergence.

Animals↗
PubMed1991

High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae.

Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.

Alleles↗
PubMed2004

Mapping domain structures in silks from insects and spiders related to protein assembly.

The exceptional solubility in vivo (20-30%, w/v) of the silk proteins of insects and spiders is dictated by both the need to produce solid fibres with a high packing fraction and the high mesogen concentration required for lyotropic liquid crystalline spinning. A further design requirement for silk proteins is a strong predominance of hydrophobic amino acid residues to provide for the hydrophobic interactions, water exclusion, and beta-crystallite formation required to produce strong insoluble threads. Thus, the domain structure of silk proteins needs to enable nanoscale phase separation to achieve high solubility of hydrophobic proteins in aqueous solutions. Additionally, silk proteins need to avoid premature precipitation as beta-sheets during storage and processing. Here we use mapping of domain types, sizes and distributions in silks to identify consistent design features that have evolved to meet these requirements. We show that silk proteins consist of conspicuously hydrophilic terminal domains flanking a very long central portion constructed from hydrophobic blocks separated by hydrophilic ones, discussing the domain structure in detail. The general rules of construction for silk proteins based on our observations should give a useful guide to the way in which Nature has solved the problem of processing hydrophobic proteins in water and how this can be copied industrially. Following these rules may also help in obtaining adequate expression, soluble products and controllable conformational switches in the production of genetically engineered or chemically synthesized silk analogues. Thus these insights have implications for structural biology and relevance to fundamental and applied questions in material science and engineering.

Animals↗
PubMed1998

Characterisation of PfSec61, a Plasmodium falciparum homologue of a component of the translocation machinery at the endoplasmic reticulum membrane of eukaryotic cells.

Plasmodium falciparum secretes several proteins that cause changes in the erythrocyte membrane enabling it to survive within red blood cells. Little is known of the mechanisms involved in the secretion and targeting of parasite polypeptides to the various cell compartments. The P. falciparum gene homologous to the mammalian Sec61alpha, gene, which encodes a component of the translocation pore in the endoplasmic reticulum of eukaryotic cells, was characterised to investigate the translocation process in the parasite. PfSec61 is present as a unique copy in the parasite genome and was mapped to chromosome 13. It encodes a 40 kDa polypeptide, as shown by immunoblotting and immunoprecipitation of [35S]methionine metabolically-labelled parasite extracts. The deduced amino acid sequence of PfSec61 is 87% similar to the mammalian polypeptide, and the two proteins give similar hydropathy plots. These results strongly suggest that PfSec61 has the same topological orientation and functional role as Sec61alpha. Anti-PfSec61 antibodies were used to investigate the cellular location and kinetics of expression of the polypeptide in the parasite. Immunofluorescence confocal microscopy showed that PfSec61 was located in the parasite cytoplasm, close to the nucleus, in a position consistent with its being in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1997

Cloning and analysis of an HMG gene from the lamprey Lampetra fluviatilis: gene duplication in vertebrate evolution.

Evolution has shaped the organisation of vertebrate genomes, including the human genome. To shed further light on genome history, we have cloned and analysed an HMG gene from lamprey, representing one of the earliest vertebrate lineages. Genes of the HMG1/2 family encode chromosomal proteins that bind DNA in a non-sequence-specific manner, and have been implicated in a variety of cellular processes dependent on chromatin structure. They are characterised by two copies of a conserved motif, the HMG box, followed by an acidic C-terminal region. We report here the cloning of a cDNA clone from the river lamprey Lampetra fluviatilis containing a gene with two HMG boxes and an acidic tail; we designate this gene LfHMG1. Molecular phylogenetic analysis shows that LfHMG1 is descended from a gene ancestral to mammalian HMG1 and HMG2. This implies that there was a duplication event in the HMG1/2 gene family, that occurred after the divergence of the jawed and jawless fishes, 450 million years ago. This conclusion supports and refines the hypothesis that there was a period of extensive gene duplication early in vertebrate evolution. We also show that the HMG1/2 family originated before the protostomes and deuterostomes diverged, over 525 million years ago.

Amino Acid Sequence↗
PubMed2002

SpADAM, a sea urchin ADAM, has conserved structure and expression.

ADAMs are multidomain cell surface proteins that function in receptor-ligand processing, cell adhesion and fusion, and signaling. SpADAM, a single copy sea urchin ADAM gene with a 3072 bp open reading frame, is expressed during embryonic and larval development. The deduced SpADAM protein is 1023 amino acids long and includes all domains characteristic of ADAMs. Northern blots reveal the presence of 4.4 and 2.3 kb SpADAM transcripts throughout development. Predominant SpADAM proteins are 131 and 95 kDa. The deduced primary structure of SpADAM is closely related to vertebrate ADAMs 12, 13, and 19. SpADAM is expressed during cleavage on blastomere surfaces, and later by vegetal plate cells, migrating secondary mesenchyme, skeletogenic mesenchyme, muscles, and neurons within the ciliated band. Apparently, the structure and types of cells in which ADAM 12/13/19 orthologues are expressed are conserved in deuterostomes.

Amino Acid Sequence↗
PubMed2003

High-density, microsphere-based fiber optic DNA microarrays.

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-microm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000-50000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 microm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA-protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

Equipment Design↗
PubMedDate not supplied

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

Adaptation, Physiological↗
PubMed2001

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

Acetyltransferases↗
PubMed2003

Using a human visual system model to optimize soft-copy mammography display: influence of MTF compensation.

RATIONALE AND OBJECTIVES: The investigators developed an efficient method for optimizing cathode ray tube (CRT) monitor performance for digital mammography, based on the correlation between the performance of human observers and the performance of a mathematical computer model of the human visual system. The investigators examined observer performance on soft-copy display of mammographic images that were either unprocessed or processed to compensate for modulation transfer function (MTF) deficiencies in the CRT display. The results were used to validate the human visual system model. MATERIALS AND METHODS: Six radiologists viewed a series of 250 mammographic images with microcalcification clusters with different contrast levels on a CRT monitor. The images were viewed twice: once without image processing and once with processing designed to compensate for MTF deficiencies in the CRT monitor. The images were analyzed with the JNDmetrix Visual Discrimination Model, which is based on the principles of just-noticeable difference measurement and frequency-channel vision modeling. Receiver operating characteristic (ROC) curves were generated for the human observers and compared statistically with the model observers' performance. RESULTS: Both human and model performance was better overall with the MTF-compensated images, especially for microcalcifications in the midlevel contrast range. There was a very high correlation between human and model observers. CONCLUSION: The use of image-processing methods to compensate for limitations in the MTF of CRT monitors can improve the detection performance of radiologists searching for microcalcifications in mammographic images, and a model based on characteristics of the human visual system can be used to predict human observer results accurately.

Computer Simulation↗
PubMed2002

Helicobacter: molecular phylogeny and the origin of gastric colonization in the genus.

The proteobacterial genus Helicobacter is composed of gastric species, all of them urease-positive, and enteric species (gastrointestinal, intestinal, hepatic, biliary), some of them urease-positive, others not. Here, we point out that the gastric species are divided in at least two phylogenetic groups, one is homogeneous, clearly separated from the enteric species, and another is forming a tight cluster within the enteric species. This feature is apparent in the phylogeny of the genus as inferred from both the 16S rRNA gene and the alpha-subunit of the urease. Our observation shows that the ability to colonize the gastric mucosa appeared more than once in the history of the genus, and suggests that acquiring this ability may be a relatively simple and punctual process, involving a limited number of genes. Such a process may be the lateral transfer acquisition of a functional copy of the gene ureI which encodes a urea channel activated at acidic pH that is essential for gastric colonization by Helicobacter pylori.

Biological Evolution↗
PubMed2000

Three ways to make a vesicle.

Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined.

Animals↗
PubMed2003

LINE-mediated retrotransposition of marked Alu sequences.

Alu elements are the most successful transposons in humans. They are 300-bp non-coding sequences transcribed by RNA polymerase III (Pol III) and are expected to retrotranspose with the aid of reverse transcriptases of cellular origin. We previously showed that human LINEs can generate cDNA copies of any mRNA transcript by means of a retroposition process involving reverse transcription and integration by the LINE-encoded endonuclease and reverse transcriptase. Here we show mobility of marked Alu sequences in human HeLa cells with the canonical features of a retrotransposition process, including splicing out of an autocatalytic intron introduced into the marked sequence, target site duplications of varying lengths and integrations into consensus A-rich sequences. We further show that the poly-A stretch at the Alu 3' end is essential for mobility, that LINEs are required for transposition and that the rate of retroposition is 100-1,000 times higher for Alu transcripts than for control mRNAs, thus accounting for the high mutational activity of these elements observed in humans.

Alu Elements↗
PubMed2001

Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1.

Classic spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of SMN1. Its product is involved in various cellular processes, including cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins, pre-mRNA processing and activation of transcription. Spinal muscular atrophy with respiratory distress (SMARD) is clinically and genetically distinct from SMA. Here we demonstrate that SMARD type 1 (SMARD1) results from mutations in the gene encoding immunoglobulin micro-binding protein 2 (IGHMBP2; on chromosome 11q13.2-q13.4). In six SMARD1 families, we detected three recessive missense mutations (exons 5, 11 and 12), two nonsense mutations (exons 2 and 5), one frameshift deletion (exon 5) and one splice donor-site mutation (intron 13). Mutations in mouse Ighmbp2 (ref. 14) have been shown to be responsible for spinal muscular atrophy in the neuromuscular degeneration (nmd) mouse, whose phenotype resembles the SMARD1 phenotype. Like the SMN1 product, IGHMBP2 colocalizes with the RNA-processing machinery in both the cytoplasm and the nucleus. Our results show that IGHMBP2 is the second gene found to be defective in spinal muscular atrophy, and indicate that IGHMBP2 and SMN share common functions important for motor neuron maintenance and integrity in mammals.

Amino Acid Sequence↗