Regions Of Sequence Homology Research Articles

DONSON: Slding in 2 the limelight

For over a decade, it has been known that yeast Sld2, Dpb11, GINS and Polε form the pre-loading complex (pre-LC), which is recruited to a CDC45-bound MCM2–7 complex by the Sld3/Sld7 heterodimer in a phospho-dependent manner. Whilst functional orthologs of Dbp11 (TOPBP1), Sld3 (TICRR) and Sld7 (MTBP) have been identified in metazoans, controversy has surrounded the identity of the Sld2 ortholog. It was originally proposed that the RECQ helicase, RECQL4, which is mutated in Rothmund-Thomson syndrome, represented the closest vertebrate ortholog of Sld2 due to a small region of sequence homology at its N-Terminus. However, there is no clear evidence that RECQL4 is required for CMG loading. Recently, new findings suggest that the functional ortholog of Sld2 is actually DONSON, a replication fork stability factor mutated in a range of neurodevelopmental disorders characterised by microcephaly, short stature and limb abnormalities. These studies show that DONSON forms a complex with TOPBP1, GINS and Polε analogous to the pre-LC in yeast, which is required to position the GINS complex on the MCM complex and initiate DNA replication. Taken together with previously published functions for DONSON, these observations indicate that DONSON plays two roles in regulating DNA replication, one in promoting replication initiation and one in stabilising the fork during elongation. Combined, these findings may help to uncover why DONSON mutations are associated with such a wide range of clinical deficits.

Genome structural variation in Escherichia coli O157:H7.

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.

Similarity of the non-amyloid-β component and C-terminal tail of monomeric and tetrameric alpha-synuclein with 14-3-3 sigma

Alpha-synuclein (αSyn) is often described as a predominantly disordered protein that has a propensity to self-assemble into toxic oligomers that are found in patients with Parkinson's and Alzheimer's diseases. αSyn's chaperone behavior and tetrameric structure are proposed to be protective against toxic oligomerization. In this paper, we extended the previously proposed similarity between αSyn and 14-3-3 proteins to the α-helical tetrameric species of αSyn in detail. 14-3-3 proteins are a family of well-folded proteins with seven human isoforms, and function in signal transduction and as molecular chaperones. We investigated protein homology, using sequence alignment, amyloid, and disorder prediction, as well as three-dimensional visualization and protein-interaction networks. Our results show sequence homology and structural similarity between the aggregation-prone non-amyloid-β component (NAC) residues Val-52 to Gly-111 in αSyn and 14-3-3 sigma residues Leu-12 to Gly-78. We identified an additional region of sequence homology in the C-terminal region of αSyn (residues Ser-129 to Asp-135) and a C-terminal loop of 14-3-3 between helix αH and αI (residues Ser-209 to Asp-215). This data indicates αSyn shares conserved domain architecture with small heat shock proteins. We show predicted regions of high amyloidogenic propensity and intrinsic structural disorder in αSyn coincide with amyloidogenic and disordered predictions for 14-3-3 proteins. The homology in the NAC region aligns with residues involved in dimer- and tetramerization of the non-amyloidogenic 14-3-3 proteins. Because 14-3-3 proteins are generally not prone to misfolding, our results lend further support to the hypothesis that the NAC region is critical to the assembly of αSyn into the non-toxic tetrameric state.

Do Arabidopsis Squamosa promoter binding Protein-Like genes act together in plant acclimation to copper or zinc deficiency?

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)‐dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu‐deficient growth conditions, Squamosa promoter binding Protein‐Like 7 (SPL7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factor SPL7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by miRNA156 with roles in plant development. SPL7 shares extended regions of sequence homology with SPL1 and SPL12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both the wild type and the spl7 single mutant under normal and Cu‐deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL7, but provided no indication for important roles of SPL1 or SPL12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii, Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn‐deficient wild type plants. Known Zn deficiency responses were unaltered in spl7, spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZnSOD activity is strongly downregulated in Zn‐deficient A. thaliana, in association with an about 94% reduction in the abundance of the CSD2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL7 and not associated with an upregulation of MIR398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR1 of Chlamydomonas. In the future, resolving how the specificity of SPL protein activation and recognition of target gene promoters is achieved will advance our understanding of the specific functions of different SPL family proteins in the regulation of either Cu deficiency responses or growth and development of land plants.

Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites.

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3’ untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern.

Molecular phylogeny and bioprospecting of Endolichenic Fungi (ELF) inhabiting in the lichens collected from a mangrove ecosystem in Sri Lanka.

Endolichenic fungi (ELF) are unexplored group of organisms as a source for the production of bioactive secondary metabolites with radical scavenging activity, antilipase and amylase inhibitory activities. Endolichenic fungi in lichens collected from mangrove or mangrove associated plants are least known for their fungal diversity and potential to produce bioactive compounds. A total of 171 ELF strains were isolated from the lichens collected from mangrove and mangrove associated plants in Puttalam lagoon. Out of this collection, 70 isolates were identified using rDNA-ITS region sequence homology to the GenBank accessions and a phylogenetic analysis was performed. Commonly isolated genera of ELF from lichens were Aspergillus, Byssochlamys, Talaromyces, Diaporthe, Phomopsis, Endomelanconiopsis, Schizophyllum, Cerrena, Trichoderma, Xylaria, Hypoxylon, Daldinia, Preussia, Sordaria, Neurospora, and Lasiodiplodia. In the present study, the effectiveness of ethyl acetate extracts of the ELF isolates were investigated against antioxidant activity, antilipase activity and α-amylase inhibition activity in in-vitro conditions. The results revealed that the extracts of Daldinia eschscholtzii, Diaporthe musigena and Sordaria sp. had the highest radical scavenging activity with smaller IC50 values (25 μg/mL to 31 μg/mL) compared to the IC50 values of BHT (76.50±1.47 μg/mL). Antilipase assay revealed that 13 extracts from ELF showed promising antiobesity activity ranged between 25% to 40%. Amylase inhibitory assay indicated that the test extracts do not contain antidiabetic secondary metabolites.

A balanced translocation disrupting SCN5A in a family with Brugada syndrome and sudden cardiac death

Brugada syndrome (BrS) is a primary arrhythmia syndrome affecting 1 in 2000 of the general population. Genetic testing identifies pathogenic variants in the sodium voltage-gated channel α-subunit 5 gene (SCN5A) in up to 25% of familial BrS. Balanced translocations, which involve the exchange of the ends of 2 different chromosomes, are found in approximately 1 in 500 people. They usually are benign and only rarely are reported to cause arrhythmogenic disorders. The purpose of this study was to identify the genetic mechanism underlying a family with BrS, sick sinus syndrome, cardiac hypertrophy, sudden cardiac death, and multiple miscarriages. We clinically evaluated family members with an electrocardiogram, 2-dimensional echocardiogram, and provocation testing with ajmaline challenge. Cytogenetic testing included karyotype and fluorescent in situ hybridization (FISH) analysis. We performed gene panel, exome, and genome sequencing analysis. Sequencing of 128 cardiac genes and exome sequencing of a family with BrS, sick sinus syndrome, cardiac hypertrophy, sudden cardiac death, and multiple miscarriages did not reveal a pathogenic variant. Karyotype and FISH analysis identified a balanced translocation breaking the SCN5A gene on chromosome 3 and the multiple chromosome maintenance 10 gene (MCM10) on chromosome 10 t(3;10)(p22.2;p13). We characterized both translocation breakpoint junctions using genome sequencing and found no regions of sequence homology. A balanced translocation breaking SCN5A is a novel mechanism underlying disease in a family with BrS, sick sinus syndrome, cardiac hypertrophy, and sudden cardiac death. Genome sequencing can identify rare chromosomal aberrations causing inherited diseases that may otherwise be missed using gene panel and exome sequencing-based approaches.

Ultradeep mapping of neuronal mitochondrial deletions in Parkinson's disease

Mitochondrial DNA (mtDNA) deletions accumulate with age in postmitotic cells and are associated with aging and neurodegenerative disorders such as Parkinson's disease. Although the exact mechanisms by which deletions form remain elusive, the dominant theory is that they arise spontaneously at microhomologous sites and undergo clonal expansion. We characterize mtDNA deletions at unprecedented resolution in individual substantia nigra neurons from individuals with Parkinson's disease, using ultradeep sequencing. We show that the number of deleted mtDNA species per neuron is substantially higher than previously reported. Moreover, each deleted mtDNA species shows significant differences in sequence composition compared with the remaining mtDNA population, which is highly consistent with independent segregation and clonal expansion. Deletion breakpoints occur consistently in regions of sequence homology, which may be direct or interrupted stretches of tandem repeats. While our results support a crucial role for misannealing in deletion generation, we find no overrepresentation of the 3′-repeat sequence, an observation that is difficult to reconcile with the current view of replication errors as the source of mtDNA deletions.

Mushroom diversity in the Biligiri rangana hills of Karnataka (India)

Evergreen forests of Biligiri rangana hills (BR hills) spread over an area of 540 sq. KM over eastern most edge of Western Ghats in Karnataka. Climatic conditions are more favorable for establishment of mushrooms and complete their life cycle. In this study, fourteen mushroom species were collected from BR hills region during monsoon (June through September 2013) with the help of Solega tribe inhabited the region since many years. Of the fourteen mushrooms, two mushrooms were identified as Ganoderma lucidum and Polyporous flabelliformis based on their phenotypic characters. The other 12 mushrooms were identified by ITS (Internal Transcribed spacer)region sequence homology as Termitomyces sp. (BRM-3)., Auricularia delicate (BRM-4), Termitomyces microcarpus (BRM-5), Amanita sp. (BRM-6), Podoscypha petalodes (BRM-7), Agaricaceae sp. (BRM-8), Macrolepiota sp.(BRM-9), Calvatia holothurioides (BRM-10), Gymnopillus crociphyllus (BRM-11), Coprinus comatus (BRM-12), Gyrodontium sacchari (BRM-13) and Clitocybeafffellea (BRM-14). Among the fourteen mushrooms, three species viz., Termito-myces species, Auricularia delicate and Termitomyces microcarpus were edible. The others were non edible/poisonous species. This study reports the diverse mushroom species as addition to biodiversity at BR hills.

25 - A Rare Case of Russell-Silver and 1p36 Deletion Syndromes

receptor gene family, which have been implicated in the etiology of autism and susceptibility to alcohol dependence, and may in tern play a role in the family’s problems with drugs and alcohol. The maternal great grandmother (I.1) did not have the duplications of 16q observed on array in the other family members. However, she was found to have a 461 kb loss on 16q21. This appears to be the same region which separates two of the duplicated regions observed in other family members. FISH analysis has determined the chromosome 16 duplicated regions have been inserted into the short arm of chromosome 4. Further molecular characterization of the duplication, deletions, and insertion are needed for a better understanding of the genomic architecture and mechanism of this complex rearrangement of chromosome 16. The complex rearrangement and copy number aberrations observed in the majority of this family appear to have begun as a unique deletion on chromosome 4p and a unique deletion of chromosome 16q21 in generation I as revealed by microarray analysis. Since at least two of her children received the insertion duplication, she most likely has a balanced version of the insertion. The complex duplication is speculated to have emerged by either nonhomologous end joining recombination (NHEJ) such as in a chromothripsis type event or non-allelic homologous recombination (NAHR) due to microhomology. NAHR mediated by regions of sequence homology at the breakpoints of the 4p deletion and the proximal and distal breakpoints of chromosome 16 segments might best explain the insertion of the 16 into chromosome 4. Further molecular characterization of the breakpoints involved will help elucidate the mechanism which produced this complex aberration.

A trivalent alphavirus virus-like replicon particle vaccine induces cell-mediated responses against conserved glycoprotein regions

Abstract Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses are highly infectious and neuroinvasive when aerosolized, making them threats for weaponization. In contrast to subcutaneous infection, studies suggest that protection from aerosol challenge is partially cell-mediated, albeit poorly understood. A trivalent (VEEV/EEEV/WEEV) virus-like replicon particle (triVRP) vaccine is protective against aerosol challenge in mice, yet the correlates of protection are unclear. To investigate, mice were vaccinated (day 0 and 28; PBS or triVRP) and spleens were harvested for analysis. Using an ex vivo splenocyte expansion assay with peptides from VEEV, EEEV and WEEV glycoprotein libraries, IFNγ-producing memory CD8+ T cells were identified. T cell-specific epitopes were mapped, and the most immunodominant were found within regions of sequence homology between strains. Thus, ongoing studies evaluate potential monovalent vaccine cross-protection, which would greatly reduce manufacturing costs. Understanding vaccine-induced responses will inform rational design and clinical evaluation.

Peptide motif analysis predicts alphaviruses as triggers for rheumatoid arthritis.

Rheumatoid arthritis (RA) develops in response to both genetic and environmental factors. The strongest genetic determinant is HLA-DR, where polymorphisms within the P4 and P6 binding pockets confer elevated risk. However, low disease concordance across monozygotic twin pairs underscores the importance of an environmental factor, probably infectious. The goal of this investigation was to predict the microorganism most likely to interact with HLA-DR to trigger RA under the molecular mimicry hypothesis. A set of 185 structural proteins from viruses or intracellular bacteria was scanned for regions of sequence homology with a collagen peptide that binds preferentially to DR4; candidates were then evaluated against a motif required for T cell cross-reactivity. The plausibility of the predicted agent was evaluated by comparison of microbial prevalence patterns to epidemiological characteristics of RA. Peptides from alphavirus capsid proteins provided the closest fit. Variations in the P6 position suggest that the HLA binding preference may vary by species, with Ross River virus, Chikungunya virus, and Mayaro virus peptides binding preferentially to DR4, and peptides from Sindbis/Ockelbo virus showing stronger affinity to DR1. The predicted HLA preference is supported by epidemiological studies of post-infection chronic arthralgia. Parallels between the cytokine profiles of RA and chronic alphavirus infection are discussed.

Development of a transformation system for the edible mushroom Grifola frondosa: Demonstrating heterologous gene expression and RNAi-mediated gene silencing

Grifola frondosa is a well-known edible mushroom that is industrially produced year-round under artificial cultivation. To improve the productivity and quality of cultivated mushrooms, transcriptomic and genomic data for G. frondosa have been used to investigate genes involved in fruiting body differentiation. However, further functional analysis of genes has been limited by the lack of specific genetic engineering tools for this species. Hence, the primary goal of this study is to establish an efficient transformation method and vector system for G. frondosa. To express transgenes efficiently, a transformation vector was constructed using homologous regulatory sequences from constitutively expressed genes identified from comprehensive gene expression profile of G. frondosa. Using this vector system, we have successfully expressed enhanced green fluorescent protein (EGFP) and firefly luciferase (Luc) in G. frondosa. Furthermore, we have tested RNA interference (RNAi)-mediated gene silencing using an RNAi vector, and showed that expression of the hairpin RNA (hpRNA) encoded by this vector can trigger downregulation of expression of Gf.HydA1, a model target hydrophobin gene from G. frondosa. Thus, the transformation system established in this study should be a useful tool for further functional analysis of other genes in G. frondosa.

Abstract 1776: Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation.

Abstract Many translocations associated with spontaneous human lymphoid malignancies are mediated by short regions of sequence homology (microhomology) at the breakpoints. The hallmark features of this error prone pathway are deletions of flanking sequence and short homologies of 1-25 bases at the junctions that serve to align the DNA ends prior to joining. In addition to its importance in the etiology of human cancers, microhomology-mediated joining or alternative NHEJ (aNHEJ) also appears to play a significant role in therapy-induced oncogenic translocations. Studies have provided compelling evidence that the DNA end-binding complex composed of Mre11 (nuclease), Rad50 (tetherer) and Nbs1 (signaler)–MRN–functions in the context of the aNHEJ pathway. In addition, in vivo and cell based studies have identified that deficiencies in components of the MRN complex reduce aNHEJ activity. However, it is not known whether the MRN complex participates in the generation of aNHEJ catalyzed translocations that can predispose to cancer. Therefore, our studies seek to better understand the contribution of Mre11/MRN to the aNHEJ pathway and thus, in generation of translocations. We have obtained mice harboring an Mre11 conditionally targeted null allele as well as an Mre11 knock-in mutation that abrogates nuclease activity. We have included mutant mice in this study that succumb to early onset Pro-B lymphomas associated with clonal translocations containing microhomologies of 2-8 nucleotides. Thus, the spontaneously arising tumors in this mutant background represent a valuable in vivo system to assess the involvement of Mre11/MRN in aNHEJ mediated translocations. We have determined that Mre11/MRN inactivation in the B-cell lineage suppresses Pro-B lymphoma in this mutant background. In future experiments we are investigating the impact of Mre11/MRN inactivation on various cancer phenotypes. Together, these studies will provide insights into the mechanisms underlying human lymphoma and may ultimately provide insights into targets for improved treatment of lymphoid malignancies. Citation Format: Cheryl J. Smith, Jeffrey Xie, David O. Ferguson, JoAnn M. Sekiguchi. Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1776. doi:10.1158/1538-7445.AM2013-1776

Improving the safety of viral DNA vaccines: development of vectors containing both 5′ and 3′ homologous regulatory sequences from non-viral origin

Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

The Dynamic Structure of the Estrogen Receptor

The estrogen receptor (ER) mediates most of the biological effects of estrogens at the level of gene regulation by interacting through its site-specific DNA and with other coregulatory proteins. In recent years, new information regarding the dynamic structural nature of ER has emerged. The physiological effects of estrogen are manifested through ER's two isoforms, ERα and ERβ. These two isoforms (ERα and ERβ) display distinct regions of sequence homology. The three-dimensional structures of the DNA-binding domain (DBD) and ligand-binding domain (LBD) have been solved, whereas no three-dimensional natively folded structure for the ER N-terminal domain (NTD) is available to date. However, insights about the structural and functional correlations regarding the ER NTD have recently emerged. In this paper, we discuss the knowledge about the structural characteristics of the ER in general and how the structural features of the two isoforms differ, and its subsequent role in gene regulation.

A Large Mutational Study in Pachyonychia Congenita

Pachyonychia congenita (PC) is a rare autosomal dominant skin disorder characterized predominantly by nail dystrophy and painful palmoplantar keratoderma. Additional clinical features include oral leukokeratosis, follicular keratosis, and cysts (steatocysts and pilosebaceous cysts). PC is due to heterozygous mutations in one of four keratin genes, namely, KRT6A, KRT6B, KRT16, or KRT17. Here, we report genetic analysis of 90 new families with PC in which we identified mutations in KRT6A, KRT6B, KRT16, or KRT17, thereby confirming their clinical diagnosis. A total of 21 previously unreported and 22 known mutations were found. Approximately half of the kindreds had mutations in KRT6A (52%), 28% had mutations in KRT16, 17% in KRT17, and 3% of families had mutations in KRT6B. Most of the mutations were heterozygous missense or small in-frame insertion/deletion mutations occurring within one of the helix boundary motif regions of the keratin polypeptide. More unusual mutations included heterozygous splice site mutations, nonsense mutations, and a 1-bp insertion mutation, leading to a frameshift and premature termination codon. This study, together with previously reported mutations, identifies mutation hotspot codons that may be useful in the development of personalized medicine for PC.

α-Enolase Autoantibodies Cross-Reactive to Viral Proteins in a Mouse Model of Biliary Atresia

Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology. The bile duct injury that occurs in patients with BA might result from a hepatobiliary viral infection followed by an autoimmune response against the bile duct epithelia. We aimed to identify autoantigens recognized by serum antibodies in the Rhesus rotavirus (RRV)-induced mouse model of BA; findings were correlated with BA in humans. Bile duct epithelial proteins were screened for their reactivity with serum antibodies from the mouse model of BA using immunoblot assays. Unique proteins that reacted with sera antibodies were identified by mass spectrometry and verified using enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Candidate autoantibodies in BA patient sera were analyzed by ELISA. A bile duct epithelial antigen that reacted strongly with serum immunoglobulin (Ig) G from the mouse model of BA was identified as α-enolase. α-Enolase autoantibody specificity was confirmed by ELISA and immunoblot analyses. Anti-RRV and anti-enolase antibodies cross-reacted with enolase and RRV proteins; we identified regions of sequence homology between RRV and enolase. Serum samples from patients with BA had increased levels of anti-enolase IgM and IgG. We have identified autoantibodies against α-enolase in a mouse model of BA (infected with RRV) and in serum samples from patients, indicating a role of humoral autoimmunity in disease pathogenesis. The cross-reactivity between an anti-enolase antibody and RRV proteins indicates that molecular mimicry might activate humoral autoimmunity in BA patients; further studies are required.

Characterization of the Bovine Pseudoautosomal Region and Comparison with Sheep, Goat, and Other Mammalian Pseudoautosomal Regions

The pseudoautosomal region (PAR) is a small region of sequence homology between mammalian X and Y chromosomes and is needed for sex chromosome segregation in male meiosis. The region, though studied as yet in only a few species, shows considerable variation in size and gene content. We have constructed a medium-density gene map for the cattle PAR and the adjacent X-specific region by isolating and mapping 18 BAC clones which contain 20 PAR- and 5 X-specific genes. One BAC clone containing TBL1XY and GPR143 spanned the recently demarcated bovine pseudoautosomal boundary (PAB). Comparing the gene map of cattle PAR with the high-resolution maps of human, horse, and dog PAR allowed to estimate that the size of cattle PAR is approximately 5–9 Mb. BAC end sequence analysis showed that there is a gradient of decreasing GC content from PARter towards the PAB which is consistent with findings in human, mouse, and horse. The 20 PAR- and 5 X-specific cattle genes were mapped also in goat and sheep, showing that PAR in the 3 species is similar in size, gene content, and gene order. For the first time the PAB was determined in goat sex chromosomes. Comparison of cattle, goat, and sheep PAR with homologous regions on human and horse X chromosomes showed a high degree of linkage conservation between all species. However, the most terminal human, horse, and dog PAR gene, PLCXD1, is X-specific in ruminants. Since the human/horse linkage group containing PLCXD1 is of ancestral origin, the location of PLCXD1 can be considered as a de novo event in ruminant sex chromosome evolution. The gene map of the cattle PAR adds to our knowledge about the comparative organization and evolution of the eutherian PAR and aids the sequencing, sequence assembly, and annotation of the terminal region of BTAXq.

Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas

Spiroplasma citri strain GII3 contains seven plasmids, pSciA and pSci1-6, that share extensive regions of sequence homology and display a mosaic gene organization. Plasmid pSci2 comprises 12 coding sequences (CDS), three of which encode polypeptides homologous to proteins Soj/ParA, involved in chromosome partitioning, and TrsE and Mob/TraG, implicated in the type IV secretion pathway. One CDS encodes the adhesin-like protein ScARP3d whereas the other eight encode polypeptides with no homology to known proteins. The pSci2 CDS pE and soj have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of S. citri 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS (pE) and its flanking intergenic regions. Shuttle (S. citri/Escherichia coli) plasmids, in which CDS pE was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein of the S. citri plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact soj gene were stably maintained, indicating that the soj-encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (S. citri/E. coli) plasmids, were shown to replicate in all S. citri strains tested regardless of whether the strain possesses endogenous plasmids, such as strain GII3, or not, such as strain R8A2. In addition, the pSci replicons were introduced efficiently into the plant-pathogenic spiroplasmas Spiroplasma kunkelii and Spiroplasma phoeniceum, the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of S. citri, the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plant-pathogenic spiroplasmas. As an example, a HA-tagged S. citri protein was expressed in S. kunkelii. Detection of pE-hybridizing sequences in various group I spiroplasma species indicated that pE replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas.