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A starch-branching enzyme gene in wheat produces alternatively spliced transcripts.

A wheat gene, denoted Sbe1, encoding a type I starch-branching enzyme (SBEI) was isolated from a genomic library and shown to comprise 14 exons distributed over a 5.7 kb DNA region. Analyses of kernel RNA by 5' rapid amplification of cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a considerable sequence variation at the 5' ends of SBEI gene transcripts. DNA sequence alignments between the 5'-RACE products and the Sbe1 genomic DNA indicated that the first two exons and first intron were differentially processed to generate three classes of the mature transcript. One form of the SBEI gene transcript in 12-day old kernels contained the exon I+II+III combination at the 5' end, whereas other forms differed by inclusion of intron 1 or exclusion of exon II sequences. RT-PCR analysis of Sbe1-uidA::nptII chimeric mRNA produced in transgenic wheat cultured cells confirmed that the isolated Sbe1 was able to produce all three forms of SBEI gene transcripts by alternative splicing of the primary mRNA. The variants of processed Sbe1 mRNA were potentially translated into N-terminal variants of the SBEI precursor with different transit peptide sequences.

1,4-alpha-Glucan Branching Enzyme↗

Phylo-VISTA: interactive visualization of multiple DNA sequence alignments.

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu

Algorithms↗

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

DNA, Complementary↗

In silico discovery of gene-coding variants in murine quantitative trait loci using strain-specific genome sequence databases.

BACKGROUND: The identification of genes underlying complex traits has been aided by quantitative trait locus (QTL) mapping approaches, which in turn have benefited from advances in mammalian genome research. Most recently, whole-genome draft sequences and assemblies have been generated for mouse strains that have been used for a large fraction of QTL mapping studies. Here we show how such strain-specific mouse genome sequence databases can be used as part of a high-throughput pipeline for the in silico discovery of gene-coding variations within murine QTLs. As a test of this approach we focused on two QTLs on mouse chromosomes 1 and 13 that are involved in physical dependence on alcohol. RESULTS: Interstrain alignment of sequences derived from the relevant mouse strain genome sequence databases for 199 QTL-localized genes spanning 210,020 base-pairs of coding sequence identified 21 genes with different coding sequences for the progenitor strains. Several of these genes, including four that exhibit strong phenotypic links to chronic alcohol withdrawal, are promising candidates to underlie these QTLs. CONCLUSIONS: This approach has wide general utility, and should be applicable to any of the several hundred mouse QTLs, encompassing over 60 different complex traits, that have been identified using strains for which relatively complete genome sequences are available.

Alcohol Withdrawal Seizures↗

Alu Overexpression Leads to an Increased Double-Stranded RNA Signature in Dermatomyositis.

OBJECTIVE: Dermatomyositis is an autoimmune condition characterized by a high interferon signature of unknown etiology. Because coding sequences constitute <1.2% of our genomes, there is a need to explore the role of the noncoding genome in disease pathogenesis. Our genomes include roughly 1.2 million Alu elements occupying approximately 10% of the genome, which can form double-stranded (ds) RNA capable of triggering MDA5 leading to interferon production. METHODS: We aligned muscle biopsy RNA sequencing data to the telomere-to-telomere reference genome and quantified short interspersed elements including Alus. Because Alus have a propensity to form dsRNA and are the major targets of both adenosine deaminase RNA specific and MDA5, we quantified adenosine to inosine (A-to-I) RNA editing, which reflects dsRNA in vivo. RESULTS: Dermatomyositis muscle (n = 39) showed a global elevation in Alu expression (including inverted-repeat Alus with high potential to form dsRNA) as well as an increased expression of unique Alu elements (n = 557, q < 0.05) compared with healthy controls (n = 34), in a pattern not seen in other myositis types (n = 81). Most (75.3%) of these Alus originated from genomic regions outside genes. A cluster of the uniquely overexpressed Alus (n = 167) correlated with interferon-stimulated genes and markers of myositis activity. Additionally, we found a uniquely expanded Alu A-to-I editome in dermatomyositis, reflecting an increase in dsRNA. Edited Alus clustered on chromosome 19, which is known to have the highest concentration of dsRNA. CONCLUSION: We hypothesize that overexpressed Alus in dermatomyositis form endogenous dsRNA that exceeds the capacity of RNA editing enzymes and triggers dsRNA sensors leading to interferon production.

Humans↗

EGenBio: a data management system for evolutionary genomics and biodiversity.

BACKGROUND: Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; http://egenbio.lsu.edu) to begin to address this. DESCRIPTION: EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. CONCLUSION: EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity.

Animals↗

How many potentially secreted proteins are contained in a bacterial genome?

Artificial neural networks were trained on the prediction of the subcellular location of bacterial proteins. A cross-validated average prediction accuracy of 93% was reached for distinction between cytoplasmic and non-cytoplasmic proteins, based on the analysis of protein amino-acid composition. Principal component analysis and self-organizing maps were used to create graphical representations of amino-acid sequence space. A clear separation of cytoplasmic, periplasmic, and extracellular proteins was observed. The neural network system was applied to predicting potentially secreted proteins in 15 complete genomes. For mesophile bacteria the predicted fractions of non-cytoplasmic proteins agree with previously published estimates, ranging between 15% and 30%. Characteristics of thermophile genomes might lead to an under-estimation of the fraction of secreted proteins by presently available prediction systems. A self-organizing map was constructed from all 15 bacterial genomes. This technique can reveal additional sequence features independent from exhaustive pair-wise sequence alignment. The Treponema pallidum and Mycobacterium tuberculosis data formed separate clusters indicating unusual characteristics of these genomes.

Amino Acid Sequence↗

The human genome browser at UCSC.

As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.

California↗

Genome annotation past, present, and future: how to define an ORF at each locus.

Driven by competition, automation, and technology, the genomics community has far exceeded its ambition to sequence the human genome by 2005. By analyzing mammalian genomes, we have shed light on the history of our DNA sequence, determined that alternatively spliced RNAs and retroposed pseudogenes are incredibly abundant, and glimpsed the apparently huge number of non-coding RNAs that play significant roles in gene regulation. Ultimately, genome science is likely to provide comprehensive catalogs of these elements. However, the methods we have been using for most of the last 10 years will not yield even one complete open reading frame (ORF) for every gene--the first plateau on the long climb toward a comprehensive catalog. These strategies--sequencing randomly selected cDNA clones, aligning protein sequences identified in other organisms, sequencing more genomes, and manual curation--will have to be supplemented by large-scale amplification and sequencing of specific predicted mRNAs. The steady improvements in gene prediction that have occurred over the last 10 years have increased the efficacy of this approach and decreased its cost. In this Perspective, I review the state of gene prediction roughly 10 years ago, summarize the progress that has been made since, argue that the primary ORF identification methods we have relied on so far are inadequate, and recommend a path toward completing the Catalog of Protein Coding Genes, Version 1.0.

Animals↗

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi.

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.

ATP-Binding Cassette Transporters↗

Long-read Sequences Mapped to a Complete Reference Genome Uncover Uncaptured Structural Variants across the Beta-globin Cluster in Africans with Sickle Cell Disease.

African genomes are marked by extensive complexity in the number and distribution of variants, yet remain under-represented in genetic databases and the human reference genome. This gap in representation limits the broad application of genomic medicine. Sickle cell disease (SCD) - one of the most common monogenic diseases - has its highest prevalence in Africa, and variation in disease severity has consistently been linked to the beta-globin locus, including levels of fetal hemoglobin (HbF). Modulation of HbF is central to current SCD gene therapies; however, the inherent complexity and variation at the locus in African genomes presents a challenge to translating these advances to Africa. Here, we align long-read single molecule sequences (LRS) targeted to the beta-globin region to the hg38 and T2T-CHM13v2 genome references in 40 individuals with SCD, predominantly recruited from three African countries. We demonstrate that the expanded T2T-CHM13v2 reference sequence at this locus reduces Structural Variant (SV) calls by 70% and uncovers uncaptured single nucleotide variants (SNVs). Across the cluster we report 343 SVs and 196 SNVs that have not been previously reported, including in LRS data from the All of Us project. By including African populations from ethnolinguistic groups that have not been previously surveyed we improve variant resolution and bolster evidence for observed variation. Finally, we identify a common &#x223c;4kb insertion locus overlapping the HBB promoter among individuals with high HbF. These results demonstrate the utility of combining a comprehensive reference genome with LRS in African populations to uncover genomic variation at disease-associated loci.

SNV↗

Molecular characterisation of SALMFamide neuropeptides in sea urchins.

The SALMFamides are a family of neuropeptides found in species belonging to the phylum Echinodermata. Members of this family have been identified in starfish (class Asteroidea) and in sea cucumbers (class Holothuroidea) but not in other echinoderms. Our aim here was to characterise SALMFamide neuropeptides in sea urchins (class Echinoidea). Radioimmunoassays for the starfish SALMFamides S1 and S2 were used to test for related peptides in whole-body acetone extracts of the sea urchin Echinus esculentus. Fractionation of extracts using high performance liquid chromatography (HPLC) revealed several peaks of SALMFamide-like immunoreactivity, with two S2-like immunoreactive peaks (3 and 4) being the most prominent. However, peak 4 could not be purified to homogeneity and although peak 3 was purified, only a partial sequence (MRYH) could be obtained. An alternative strategy for identification of echinoid SALMFamides was provided by sequencing the genome of the sea urchin Strongylocentrotus purpuratus. Analysis of whole-genome shotgun sequence data using the Basic Local Alignment Search Tool (BLAST) identified a contig (347664) that contains a coding region for seven putative SALMFamide neuropeptides (PPVTTRSKFTFamide, DAYSAFSFamide, GMSAFSFamide, AQPSFAFamide, GLMPSFAFamide, PHGGSAFVFamide and GDLAFAFamide), which we have named SpurS1-SpurS7, respectively. Three of these peptides (SpurS1-3) have the C-terminal sequences TFamide or SFamide, which are identical or similar to the C-terminal region of the starfish SALMFamide S2. This may explain the occurrence of several S2-like immunoreactive peptides in extracts of Echinus esculentus. Detailed analysis of the sequence of contig 347664 indicated that the SALMFamide gene in Strongylocentrotus purpuratus comprises two exons, with the first exon encoding a signal peptide sequence and the second exon encoding SpurS1-SpurS7. Characterisation of this gene is important because it is the first echinoderm neuropeptide precursor sequence to be identified and, more specifically, it provides our first insight into the structure and organisation of a SALMFamide gene in an echinoderm. In particular, it has revealed a hitherto unknown complexity in the diversity of SALMFamide neuropeptides that may occur in an echinoderm species because all previous studies, which relied on peptide purification and sequencing, revealed only two SALMFamide neuropeptides in each species examined. It now remains to be established whether or not the occurrence of more than two SALMFamides in Strongylocentrotus purpuratus is a feature that is peculiar to this species and to echinoids in general or is more widespread across the phylum Echinodermata. Identification of SpurS1-SpurS7 provides the basis for comparative analysis of the physiological actions of these peptides in sea urchins and for exploitation of the sea urchin genome sequence to identify the receptor(s) that mediate effects of SALMFamides in echinoderms.

Amino Acid Sequence↗

GRASP-DNA: a web application to screen prokaryotic genomes for specific DNA-binding sites and repeat motifs.

The ability to control multiple genes at the transcriptional level often relies on the existence of short stretches of well-defined DNA sequences, to which regulatory proteins and transcription factors bind. In this article we present a freely accessible web-based application (GRASP-DNA), that can be used to screen prokaryotic genomes for putative DNA-binding sites of a particular transcription factor or DNA-binding molecule. This application utilizes existing theories, such as information and statistical-mechanical theories, for the calculation of positive weight matrices generated from block aligned binding sites. Using these position weight matrices entire prokaryotic genomes are screened to identify sites that display a high level of sequence similarity to existing binding sites. This application can be used in combination with high-throughput technologies for gene expression analysis and binding site characterization to assist in the elucidation of global regulatory networks.

Base Sequence↗

Sequence and comparative analysis of the maize NB mitochondrial genome.

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.

Base Sequence↗

cDNA sequence and tissue expression of Fugu rubripes prion protein-like: a candidate for the teleost orthologue of tetrapod PrPs.

We report the isolation and characterization of a cDNA coding for Fugu rubripes prion protein (PrP)-like of 180 amino acids which includes the PrP-conserved hydrophobic region homologous to that of Xenopus PrP. In addition to the hydrophobic region, Fugu PrP-like has several features common to PrPs, such as a signal sequence, a basic nature (pI 9.7) and a single intron in the 5' untranslated region. A possible glycosyl phosphatidylinositol (GPI) anchor site also exists in PrP-like. In expression analysis, PrP-like mRNA was detected in retina, skin, and brain, all of which express PrP mRNA in mammals. In a genome fragment clone (T002589, 31945 bp) sequenced by the Fugu Genomics Project, PrP-like located between KIAA0168 and SLC231A homologues. In human chromosome 20p13, PrP, Doppel, KIAA0168, and SLC231A align in this order. The close gene arrangement between the Fugu and human genomes suggests that Fugu PrP-like is a real orthologue of human PrP. However, Fugu PrP-like does not possess tandem repeats or a region with two glycosylation sites and a disulphide bridge. We do not declare that the cloned Fugu PrP-like represents fish PrP due to structural inconsistency, but believe that it will offer new insights into the evolution of PrPs from fish to tetrapods.

Amino Acid Sequence↗

Nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptides.

A region of the feline calicivirus (FCV) genome was sequenced which encodes polypeptides that are similar by amino acid alignment analysis to several picornavirus polypeptides. These polypeptides include the 2C polypeptide, the 3C cysteine protease and the 3D RNA-dependent RNA polymerase. The 2C-like region of the FCV genome encodes a GxxGxGKT nucleotide binding motif as well as amino acids which have been shown to be conserved in the picornavirus 2C polypeptides. The FCV RNA-dependent RNA polymerase also shows regions of similarity with picornavirus RNA polymerase sequences including the GDD sequence which is thought to be in or near the active site of the polymerase. The FCV cysteine protease-like sequences have the lowest degree of similarity with picornavirus cysteine proteases of the three regions aligned. However, the cysteine and histidine residues thought to be in the active site of the protease are present and are surrounded by amino acids conserved in the picornavirus cysteine proteases. The order of the polypeptides encoded in the FCV genome is the same as in the picornaviruses with the RNA-dependent RNA polymerase being located at the C-terminus of the FCV polyprotein. However, there is an approximately 40,000 dalton region between the FCV 2C- and the cysteine protease-like polypeptides which has no similarity to any known picornavirus protein. A striking difference between the organization of these sequences in FCV and the picornaviruses is that in the FCV genome, these non-structural polypeptides are encoded near the 5' end of the genomic RNA. Termination of the reading frame encoding these polypeptides occurs approximately 2400 bases from the 3' end of the genomic RNA as compared to 71 bases in the poliovirus genomic RNA.

Amino Acid Sequence↗

Identification of candidate disease genes by EST alignments, synteny, and expression and verification of Ensembl genes on rat chromosome 1q43-54.

We aligned Incyte ESTs and publicly available sequences to the rat genome and analyzed rat chromosome 1q43-54, a region in which several quantitative trait loci (QTLs) have been identified, including renal disease, diabetes, hypertension, body weight, and encephalomyelitis. Within this region, which contains 255 Ensembl gene predictions, the aligned sequences clustered into 568 Incyte genes and gene fragments. Of the Incyte genes, 261 (46%) overlapped 184 (72%) of the Ensembl gene predictions, whereas 307 were unique to Incyte. The rat-to-human syntenic map displays rearrangement of this region on rat chr. 1 onto human chromosomes 9 and 10. The mapping of corresponding human disease phenotypes to either one of these chromosomes has allowed us to focus in on genes associated with disease phenotypes. As an example, we have used the syntenic information for the rat Rf-1 disease region and the orthologous human ESRD disease region to reduce the size of the original rat QTL to only 11.5 Mb. Using the syntenic information in combination with expression data from ESTs and microarrays, we have selected a set of 66 candidate disease genes for Rf-1. The combination of the results from these different analyses represents a powerful approach for narrowing the number of genes that could play a role in the development of complex diseases.

Animals↗

Comparison of Salmonella enterica serovar Typhimurium LT2 and non-LT2 salmonella genomic sequences, and genotyping of salmonellae by using PCR.

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.

Bacterial Typing Techniques↗