Lone Bruhn Madsen

Insights into the Evolution of Longevity from the Bowhead Whale Genome

Summary The bowhead whale (Balaena mysticetus) is estimated to live over 200 years and is possibly the longest-living mammal. These animals should possess protective molecular adaptations relevant to age-related diseases, particularly cancer. Here, we report the sequencing and comparative analysis of the bowhead whale genome and two transcriptomes from different populations. Our analysis identifies genes under positive selection and bowhead-specific mutations in genes linked to cancer and aging. In addition, we identify gene gain and loss involving genes associated with DNA repair, cell-cycle regulation, cancer, and aging. Our results expand our understanding of the evolution of mammalian longevity and suggest possible players involved in adaptive genetic changes conferring cancer resistance. We also found potentially relevant changes in genes related to additional processes, including thermoregulation, sensory perception, dietary adaptations, and immune response. Our data are made available online (http://www.bowhead-whale.org) to facilitate research in this long-lived species.

A robust linkage map of the porcine autosomes based on gene-associated SNPs

BackgroundGenetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences.ResultsLinkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations.ConclusionThe linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.

Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain

BackgroundThe transmembrane presenilin (PSEN) proteins, PSEN1 and PSEN2, have been proposed to be the catalytic components of the γ-secretase protein complex, which is an intramembranous multimeric protease involved in development, cell regulatory processes, and neurodegeneration in Alzheimers disease. Here we describe the sequencing, chromosomal mapping, and polymorphism analysis of PSEN1 and PSEN2 in the domestic pig (Sus scrofa domesticus).ResultsThe porcine presenilin proteins showed a high degree of homology over their entire sequences to the PSENs from mouse, bovine, and human. PSEN1 and PSEN2 transcription was examined during prenatal development of the brain stem, hippocampus, cortex, basal ganglia, and cerebellum at embryonic days 60, 80, 100, and 114, which revealed distinct temporal- and tissue-specific expression profiles. Furthermore, immunohistochemical analysis of PSEN1 and PSEN2 showed similar localization of the proteins predominantly in neuronal cells in all examined brain areas.ConclusionThe data provide evidence for structural and functional conservation of PSENs in mammalian lineages, and may suggest that the high sequence similarity and colocalization of PSEN1 and PSEN2 in brain tissue reflect a certain degree of functional redundancy. The data show that pigs may provide a new animal model for detailed analysis of the developmental functions of the PSENs.

Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na+/K+-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na+ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Porcine γ-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression

The γ-synuclein protein is involved in breast carcinogenesis and has also been implicated in other forms of cancer and in ocular diseases. Furthermore, γ-synuclein is believed to have a role in certain neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. This work reports the cloning and characterization of the porcine (Sus scrofa) γ-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids which shows a high similarity to bovine (90%), human (87%) and mouse (83%) γ-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex and pituitary gland. Expression analysis also showed that porcine SNCG transcripts could be detected in different brain regions during early stages of embryo development. The porcine SNCG orthologue was mapped to chromosome 14q25–q29. The distribution of recombinant porcine γ-synuclein was studied in three different transfected cell lines and the protein was found to be predominantly localized in the cytoplasm.

Porcine DJ-1: cloning of PARK7 cDNA, sequence comparison, expression analysis and chromosomal localization

The PARK7 gene encodes a protein, DJ-1, with several functions such as protection of cells from oxidative stress, sperm maturation and fertilization and chaperone activity. Mutations in the PARK7 gene are associated with autosomal recessive early-onset Parkinson’s disease (Parkinsonism). This work reports the cloning and analysis of the porcine (Sus scrofa) homologue of DJ-1. The porcine PARK7 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine PARK7 cDNA (SsPARK7) encodes a protein of 189 amino acids which shows a very high similarity to bovine (97%), to human (96%) and to canine (95%) DJ-1. Protein structure comparison of human and porcine DJ-1 sequences revealed that amino acid changes were few between the two species and not likely to alter DJ-1 structure and function. Quantitative real-time RT-PCR detection exhibited SsPARK7 mRNA expression in all analyzed porcine tissues, although at different levels. Furthermore, expression analysis showed that SsPARK7 transcripts could be detected early in embryo development in different brain regions. The PARK7 gene was demonstrated to be located on porcine chromosome 6. Single-nucleotide polymorphism (SNP) analysis revealed one SNP in the porcine PARK7 gene, giving rise to a silent mutation in exon 6.

Molecular cloning, characterization and developmental expression of porcine β-synuclein

The synuclein family includes three known proteins: α-synuclein, β-synuclein and γ-synuclein. β-Synuclein inhibits the aggregation of α-synuclein, a protein involved in Parkinson’s disease. We have cloned and characterized the cDNA sequence for porcine β-synuclein (SNCB) from pig cerebellum using RT-PCR. Expression analysis by quantitative RT-PCR demonstrated that SNCB transcripts were highly abundant in brain tissues. SNCB mRNA was also detected early in embryogenesis and significant increases in transcript levels were observed in several brain tissues during embryo development. Radiation hybrid mapping data indicate that the porcine SNCB maps to the q arm of chromosome 2 (2q21-22). The subcellular localization of recombinant porcine β-synuclein was determined in three different cell types and shown to be cytoplasmic.

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain

The porcine COL10A1 gene, encoding the α1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.

The first draft reference genome of the American mink ( Neovison vison )

The American mink (Neovison vison) is a semiaquatic species of mustelid native to North America. It’s an important animal for the fur industry. Many efforts have been made to locate genes influencing fur quality and color, but this search has been impeded by the lack of a reference genome. Here we present the first draft genome of mink. In our study, two mink individuals were sequenced by Illumina sequencing with 797 Gb sequence generated. Assembly yielded 7,175 scaffolds with an N50 of 6.3 Mb and length of 2.4 Gb including gaps. Repeat sequences constitute around 31% of the genome, which is lower than for dog and cat genomes. The alignments of mink, ferret and dog genomes help to illustrate the chromosomes rearrangement. Gene annotation identified 21,053 protein-coding sequences present in mink genome. The reference genome’s structure is consistent with the microsatellite-based genetic map. Mapping of well-studied genes known to be involved in coat quality and coat color, and previously located fur quality QTL provide new knowledge about putative candidate genes for fur traits. The draft genome shows great potential to facilitate genomic research towards improved breeding for high fur quality animals and strengthen our understanding on evolution of Carnivora.

Splicing variants of porcine synphilin-1

Parkinsons disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinsons disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.