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PubMed2001

Keratin K6irs is specific to the inner root sheath of hair follicles in mice and humans.

BACKGROUND: Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified. OBJECTIVES: To characterize a novel type II keratin in mice and humans. METHODS: Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)-PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence. RESULTS: Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT-PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically. CONCLUSIONS: This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome.

Amino Acid Sequence↗

PubMed2026

Unravelling the genomic potential of sponge-associated Streptomyces sp. BLC 17-3 from Indonesia for mannooligosaccharide production.

This research aims to show the promising capacity of Streptomyces sp. BLC 17-3 to produce high β-mannanase enzymes and generate mannooligosaccharide (MOS) such as mannobiose, mannotriose, mannotetraose and mannopentaose when exposed to mannan polymers. Streptomyces sp. BLC 17-3 was isolated from the sponge (Rhabdastrella globostellata) Put4 obtained from the marine waters of Putus Island in Bitung, North Sulawesi, Indonesia. The characterization results showed that the peak enzyme activity was achieved at 50 mM sodium acetate, 6.0 pH, and 60 °C temperature on the seventh day of production with a value of 155.77 ± 3.21 U/mL. The SDS-PAGE and zymograms also showed that the size of the enzyme molecule was approximately ±34.8-49.1 kDa. Moreover, whole-genome sequencing was conducted to identify the genetic basis of MOS-synthesizing capabilities in the selected strain, followed by functional annotation of genes encoding mannan degradation and associated functions. The results showed an 8,248,862 Mb complete draft genome of the strain which comprised 111 predicted gene models. Gene annotation also provided important information about the location and function of protein-encoding genes. A total of 6 mannan degradation-related genes encoding mannanase-related metabolism were identified and the three-dimensional structures were predicted using AlphaFold 3. This characterization and modeling further enhanced the bioprospecting and development of this strain which exhibited efficient mannose metabolism. The results showed Streptomyces sp. BLC 17-3 as a promising microorganism for the future bioproduction of MOS which were discovered to have the capability of serving as a potential prebiotic substance to enhance digestion and promote health.

Bioprospecting↗

PubMed2006

Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays.

BACKGROUND: Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. RESULTS: Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. CONCLUSION: We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research.

Aeromonas salmonicida↗

PubMed2003

A comparative analysis of HGSC and Celera human genome assemblies and gene sets.

MOTIVATION: Since the simultaneous publication of the human genome assembly by the International Human Genome Sequencing Consortium (HGSC) and Celera Genomics, several comparisons have been made of various aspects of these two assemblies. In this work, we set out to provide a more comprehensive comparative analysis of the two assemblies and their associated gene sets. RESULTS: The local sequence content for both draft genome assemblies has been similar since the early releases, however it took a year for the quality of the Celera assembly to approach that of HGSC, suggesting an advantage of HGSC's hierarchical shotgun (HS) sequencing strategy over Celera's whole genome shotgun (WGS) approach. While similar numbers of ab initio predicted genes can be derived from both assemblies, Celera's Otto approach consistently generated larger, more varied gene sets than the Ensembl gene build system. The presence of a non-overlapping gene set has persisted with successive data releases from both groups. Since most of the unique genes from either genome assembly could be mapped back to the other assembly, we conclude that the gene set discrepancies do not reflect differences in local sequence content but rather in the assemblies and especially the different gene-prediction methodologies.

Databases, Protein↗

PubMed2004

Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes.

With about 24,000 extant species, teleosts are the largest group of vertebrates. They constitute more than 99% of the ray-finned fishes (Actinopterygii) that diverged from the lobe-finned fish lineage (Sarcopterygii) about 450 MYA. Although the role of genome duplication in the evolution of vertebrates is now established, its role in structuring the teleost genomes has been controversial. At least two hypotheses have been proposed: a whole-genome duplication in an ancient ray-finned fish and independent gene duplications in different lineages. These hypotheses are, however, based on small data sets and lack adequate statistical and phylogenetic support. In this study, we have made a systematic comparison of the draft genome sequences of Fugu and humans to identify paralogous chromosomal regions ("paralogons") in the Fugu that arose in the ray-finned fish lineage ("fish-specific"). We identified duplicate genes in the Fugu by phylogenetic analyses of the Fugu, human, and invertebrate sequences. Our analyses provide evidence for 425 fish-specific duplicate genes in the Fugu and show that at least 6.6% of the genome is represented by fish-specific paralogons. We estimated the ages of Fugu duplicate genes and paralogons using the molecular clock. Remarkably, the ages of duplicate genes and paralogons are clustered, with a peak around 350 MYA. These data strongly suggest a whole-genome duplication event early during the evolution of ray-finned fishes, probably before the origin of teleosts.

Animals↗

PubMed2004

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis.

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.

Animals↗

PubMed2003

GeneSeqer@PlantGDB: Gene structure prediction in plant genomes.

The GeneSeqer@PlantGDB Web server (http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi) provides a gene structure prediction tool tailored for applications to plant genomic sequences. Predictions are based on spliced alignment with source-native ESTs and full-length cDNAs or non-native probes derived from putative homologous genes. The tool is illustrated with applications to refinement of current gene structure annotation and de novo annotation of draft genomic sequences. The service should facilitate expert annotation as a community effort by providing convenient access to all public plant sequences via the PlantGDB database, a simple four-step protocol for spliced alignment and visually appealing displays of the predicted gene structures in addition to detailed sequence alignments.

Arabidopsis↗

PubMed2003

The ascidian tadpole larva: comparative molecular development and genomics.

Evolution is of interest not only to developmental biology but also to genetics and genomics. We are witnessing a new era in which evolution, development, genetics and genomics are merging to form a new discipline, a good example of which is the study of the origin and evolution of the chordates. Recent studies on the formation of the notochord and the dorsal neural tube in the increasingly famous Ciona intestinalis tadpole larva, and the availability of its draft genome, show how the combination of comparative molecular development and evolutionary genomics might help us to better understand our chordate ancestor.

Animals↗

PubMed2004

Complex SNP-related sequence variation in segmental genome duplications.

There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.

Evolution, Molecular↗

PubMed2004

Bovine genomics update: making a cow jump over the moon.

Recent research in bovine genetics has focused on characterization of the biological differences underlying phenotypic variation for qualitative and quantitative traits of economic value in existing cattle populations. The much-anticipated benefits of DNA-based tools to routinely guide selection decisions for more efficient genetic gain and widened profit margins have not been fully met since the origin of this premise over two decades ago. However, the impending release of a high-quality draft genome sequence in 2005 should mark a turning point in these efforts. The following discussion summarizes how the bovine genetics research community has positioned itself to fully utilize a genome sequence resource and frames how genome sequence information can not only be applied to better implement marker-assisted selection, but also address rising consumer concerns relative to animal well-being and food safety.

Animals↗

PubMed2005

Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis.

The Sox and Forkhead (Fox) gene families are comprised of transcription factors that play important roles in a variety of developmental processes, including germ layer specification, gastrulation, cell fate determination, and morphogenesis. Both the Sox and Fox gene families are divided into subgroups based on the amino acid sequence of their respective DNA-binding domains, the high-mobility group (HMG) box (Sox genes) or Forkhead domain (Fox genes). Utilizing the draft genome sequence of the cnidarian Nematostella vectensis, we examined the genomic complement of Sox and Fox genes in this organism to gain insight into the nature of these gene families in a basal metazoan. We identified 14 Sox genes and 15 Fox genes in Nematostella and conducted a Bayesian phylogenetic analysis comparing HMG box and Forkhead domain sequences from Nematostella with diverse taxa. We found that the majority of bilaterian Sox groups have clear Nematostella orthologs, while only a minority of Fox groups are represented, suggesting that the evolutionary pressures driving the diversification of these gene families may be distinct from one another. In addition, we examined the expression of a subset of these genes during development in Nematostella and found that some of these genes are expressed in patterns consistent with roles in germ layer specification and the regulation of cellular behaviors important for gastrulation. The diversity of expression patterns among members of these gene families in Nematostella reinforces the notion that despite their relatively simple morphology, cnidarians possess much of the molecular complexity observed in bilaterian taxa.

Amino Acid Sequence↗

PubMed2003

Evolutionary conservation of CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human.

The CCND1-ORAOV1-FGF19-FGF4-FGF3-FLJ10261-FADD-PPFIA1-EMS1 locus on human chromosome 11q13 is frequently amplified in esophageal cancer, breast cancer, and bladder tumors. FGF19, FGF4 and FGF3 genes are implicated in embryogenesis and carcinogenesis. We proposed in 2002 the hypothesis that mouse Fgf15 might be the ortholog of human FGF19 based on comparative genomics. Here, we identified zebrafish fgf19 and oraov1 genes by using bioinformatics to demonstrate the hypothesis. Zebrafish fgf19 gene, consisting of three exons, was located around nucleotide position 121802-124963 of zebrafish genome draft sequence AL929586.12 in the reverse orientation. Zebrafish fgf19 (209 aa) was more homologous to chicken fgf19 and human FGF19 than to rodent Fgf15. Zebrafish oraov1 gene, consisting of five exons, was located around nucleotide position 112172-115838 of AL929586.12 in the reverse orientation. Zebrafish oraov1 protein (141 aa) was more homologous to human ORAOV1 than to rodent Oraov1. The CCND1-ORAOV1-FGF19-FGF4 locus was well conserved between human and zebrafish genomes in the order of genes, in the direction of genes, and in the exon-intron structure. Rat Ccnd1-Oraov1-Fgf15-Fgf4 locus was synthenic to mouse Ccnd1-Oraov1 (also known as 2210010N10Rik)-Fgf15-Fgf4 locus. Fgf15, homologous to human FGF19 and zebrafish fgf19, was located on the synthenic locus of human FGF19 and zebrafish fgf19 within rodent genomes. Based on the evolutionary conservation of the CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human, it was concluded that Fgf15 gene is the rodent ortholog of human FGF19 gene.

Amino Acid Sequence↗

PubMed2006

An integrated BAC and genome sequence physical map of Phytophthora sojae.

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.

Chromosomes, Artificial, Bacterial↗

PubMed2003

Potential for retroposition by old Alu subfamilies.

Alu elements sharing sequence characteristics of the "old" subfamilies are thought to currently be retrotranspositionally inactive. We analyzed one of these old subfamilies of Alu elements, Sx, for sequence conservation relative to the consensus and the length of the "A-tail" as parameters to define the presence of potential Alu Sx source genes in the human genome. Sequence identity to the left half or the right half of the Alu Sx consensus sequence was evaluated for 4424 complete elements obtained from the human genome draft sequence. A small subset of Alu Sx left halves were found to be more conserved than any of the Alu Sx right halves. Selection for promoter function in active elements may explain the slightly higher conservation of the left half. In order to determine whether this sequence identity was the result of recent activity, or simply sequence conservation for older elements, PCR amplification of some of the loci containing Sx elements with conserved left/right halves from different primate genomes was carried out. Several of these Sx Alus were found to have amplified at a later evolutionary period (<35 mya) than expected based on previous studies of Sx elements. Analysis of "A-tail" length, a feature correlated with current retroposition activity, varied between Alu Sx element loci in different primates, where the length increased in specific Alu elements in the human genome. The presence of few conserved Alu Sx elements and the dynamic expansion/contraction of the A-tail suggests that some of these older subfamilies may still be active at very low levels or in a few individuals.

Alu Elements↗

PubMed2004

Analysis of the human Alu Ya-lineage.

The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.

Alu Elements↗

PubMed2004

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ.

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

Amino Acid Sequence↗

PubMed2025

Sawfish: improving long-read structural variant discovery and genotyping with local haplotype modeling.

MOTIVATION: Structural variants (SVs) play an important role in evolutionary and functional genomics but are challenging to characterize. High-accuracy, long-read sequencing can substantially improve SV characterization when coupled with effective calling methods. While state-of-the-art long-read SV callers are highly accurate, further improvements are achievable by systematically modeling local haplotypes during SV discovery and genotyping. RESULTS: We describe sawfish, an SV caller for mapped high-quality long reads incorporating systematic SV haplotype modeling to improve accuracy and resolution. Assessment against the draft Genome in a Bottle (GIAB) SV benchmark from the T2T-HG002-Q100 diploid assembly shows that sawfish has the highest accuracy among state-of-the-art long-read SV callers across every tested SV size group. Additionally, sawfish maintains the highest accuracy at every tested depth level from 10- to 32-fold coverage, such that other callers required at least 30-fold coverage to match sawfish accuracy at 15-fold coverage. Sawfish also shows the highest accuracy in the GIAB challenging medically relevant genes benchmark, demonstrating improvements in both comprehensive and medically relevant contexts.When joint-genotyping seven samples from CEPH-1463, sawfish has over 9000 more pedigree-concordant calls than other state-of-the-art SV callers, with the highest proportion of concordant SVs (81%). Sawfish's quality model enables selection for an even higher proportion of concordant SVs (88%), while still calling nearly 5000 more pedigree-concordant SVs than other callers. These results demonstrate that sawfish improves on the state-of-the-art for long-read SV calling accuracy across both individual and joint-sample analyses. AVAILABILITY AND IMPLEMENTATION: Sawfish source code, pre-compiled Linux binaries, and documentation are released on GitHub: https://github.com/PacificBiosciences/sawfish.

Haplotypes↗

PubMed2004

Primitive complement system of invertebrates.

Most components of the human complement system have unmistakable domain architectures, making evolutionary tracing feasible. In contrast to the major genes of the adaptive immune system, which are present only in jawed vertebrates, complement component genes with unique domain structures are present not only in jawed vertebrates but also in jawless fish and non-vertebrate deuterostomes. Recent progress in genome analysis in several eukaryotes, occupying the phylogenetically critical positions, showed that most individual domains found in the complement components are metazoa specific, being found both in deuterostomes and in protostomes but not in yeast or plant. However, unique domain architecture of complement components is not present in protostomes, suggesting that the complement system has been established in the deuterostome lineage not by invention of new domains but by innovation of unique combination of the pre-existing domains. The recently assembled Ciona intestinalis draft genome contained the most modular complement genes, except for factor I. However, some possible C. intestinalis complement components show critical structural divergence from the mammalian counterparts, casting doubt on their mutual interaction. Thus, another integrative step seems to have been required to establish the modern complement system of higher vertebrates.

Animals↗