Gene conversion causing human inherited disease: Evidence for involvement of non‐B‐DNA‐forming sequences and recombination‐promoting motifs in DNA breakage and repair

A variety of DNA sequence motifs including inverted repeats, minisatellites, and the χ recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)‐ and CpG‐rich regions and that sequences with the potential to form non‐B‐DNA structures, and which may be involved in the generation of double‐strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the χ‐element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination‐inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome. Hum Mutat 30:1–10, 2009. © 2009 Wiley‐Liss, Inc.     

A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, we have identified a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome. Detailed sequence analysis indicates that this domain contains no centromeric alpha-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human alpha-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed.     

A new Ig-superfamily member,molluscan defence molecule (MDM) from Lymnaea stagnalis,is down-regulated during parasitosis

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to ～21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper functioning of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling of non-self recognition.     

Molecular Basis for Antigenic Variation of a Protective Strain-Specific Antigen of Ehrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, has recently been isolated from many vaccinated horses with typical clinical signs of the disease. The heterogeneity of the E. risticii isolates obtained from the vaccinated horses necessitates the identification of the molecular basis of strain variations to elucidate the vaccine failure and to aid in the development of an efficient vaccine against this disease. As an attempt, two major cross-reacting surface antigen genes of 50- and 85-kDa antigens, present separately in strains 25-D (isolated in 1984) and 90-12 (isolated in 1990 from a vaccinated horse), respectively, were cloned and sequenced. A comparative sequence analysis revealed differences and similarities between these two antigens with strain-specific sizes (SSA). The 2.5- and 1.6-kb genes coding for the 85- and 50-kDa proteins, respectively, contained many different tandem repeats. The identical repeat motifs were more frequent in the middle of both genes, but the numbers and positions of the repeats were altogether different in the genes. Many of these direct repeats of both genes had exact sequence homology and coded for the same amino acids. The homology of the 5′- and 3′-flanking regions of the two genes was greater than that of the regions in the central part of the genes. A comparative analysis of the deduced amino acid sequences of these two antigen genes indicated eight common domains, which were designated identical domains. Although the sequence homologies of these identical domains were the same, the positions of the domains in their respective strains were completely different. This finding might be one of the bases of antigenic variation between the strains. In addition, there were a few unique regions in both antigen genes where no sequence homology existed. These specific regions were designated unique domains. The 50-kDa protein had two such unique domains, and the 85-kDa protein had six such unique domains. The presence of such unique domains contributed to the large size variation of these SSA. The cross-reactivity of recombinant proteins confirmed the presence of conserved epitopes between these two antigens. The SSA have been determined to be apparent protective antigens of E. risticii.     

Phylogenetic Analysis and Homologies of the Replicase of Tomato Leaf Curl Geminiviruses: Implications for Obtaining Pathogen Derived Resistance

Geminiviruses encode a replication initiator protein, Rep, which binds to iterated DNA motifs functioning as essential elements for virus specific replication. Rep protein gene of three isolates of whitefly transmitted geminivirus causing leaf curl disease of tomato in India were amplified, cloned and sequenced. Nucleotide sequence and the derived amino acid sequence for the replicase gene of these isolates was determined and included in an analysis with the published sequences. Phylogenetic relationship clearly indicates two subsets, one belonging to Tomato leaf curl virus (ToLCV) having bipartite genome and the other having the monopartite genome. Comparisons of various functional domains and motifs involved in specificity determinants, DNA-binding and catalysis were discussed. The implications of the sequence analysis were discussed with respect to the strategy for the generation of engineered resistance having wide spectrum applications.     

A simple electronic tool for assessing amino acid sequence polymorphisms within exon-2 of HLA-DPB1 alleles

In November 2014, the OPTN/UNOS Board of Directors mandated that HLA-DPB1 typing be performed for all deceased donors. Currently, there are over 1,000 known HLA DPB1 alleles, yet fewer than 30 are represented on commonly used single antigen bead (SAB) solid phase antibody assays. Moreover, the official World Health Organization (WHO) nomenclature for the DPB1 locus does not permit assessment of structural relationships between alleles based on their names. Thus, for donor DPB1 alleles lacking a corresponding SAB, determining the compatibility between a donor-recipient pair when the recipient possesses DPB1 antibodies currently requires the use of manual sequence alignments. Multiple studies have reported that DPB1 alleles can be classified into serological-defined categories based on shared protein sequence motifs residing in distinct hypervariable regions. To date, six such motifs have been recognized. To address this problem, we developed a computer-assisted tool to compare donor and recipient DPB1 allele sequences, specifically those defined by DPB1 hypervariable region motifs located in exon 2 (http://dpreport.hlatools.org). This tool quickly identifies mismatched DPB1 motifs, and easily permits the identification of motif-based donor-specific antibodies (DSA) to DPB1.     

Measuring single-cell protein secretion in immunology: Technologies,advances, and applications

The dynamics, nature, strength, and ultimately protective capabilities of an active immune response are determined by the extracellular constitution and concentration of various soluble factors. Generated effector cells secrete such mediators, including antibodies, chemo- and cytokines to achieve functionality. These secreted factors organize the individual immune cells into functional tissues, initiate, orchestrate, and regulate the immune response. Therefore, a single-cell resolved analysis of protein secretion is a valuable tool for studying the heterogeneity and functionality of immune cells. This review aims to provide a comparative overview of various methods to characterize immune reactions by measuring single-cell protein secretion. Spot-based and cytometry-based assays, such as ELISpot and flow cytometry, respectively, are well-established methods applied in basic research and clinical settings. Emerging novel technologies, such as microfluidic platforms, offer new ways to measure and exploit protein secretion in immune reactions. Further technological advances will allow the deciphering of protein secretion in immunological responses with unprecedented detail, linking secretion to functionality. Here, we summarize the development and recent advances of tools that allow the analysis of protein secretion at the single-cell level, and discuss and contrast their applications within immunology.     

The repetitive landscape of the chicken genome

Cot-based cloning and sequencing (CBCS) is a powerful tool for isolating and characterizing the various repetitive components of any genome, combining the established principles of DNA reassociation kinetics with high-throughput sequencing. CBCS was used to generate sequence libraries representing the high, middle, and low-copy fractions of the chicken genome. Sequencing high-copy DNA of chicken to about 2.7 x coverage of its estimated sequence complexity led to the initial identification of several new repeat families, which were then used for a survey of the newly released first draft of the complete chicken genome. The analysis provided insight into the diversity and biology of known repeat structures such as CR1 and CNM, for which only limited sequence data had previously been available. Cot sequence data also resulted in the identification of four novel repeats (Birddawg, Hitchcock, Kronos, and Soprano), two new subfamilies of CR1 repeats, and many elements absent from the chicken genome assembly. Multiple autonomous elements were found for a novel Mariner-like transposon, Galluhop, in addition to nonautonomous deletion derivatives. Phylogenetic analysis of the high-copy repeats CR1, Galluhop, and Birddawg provided insight into two distinct genome dispersion strategies. This study also exemplifies the power of the CBCS method to create representative databases for the repetitive fractions of genomes for which only limited sequence data is available.     

Simple method for determination of the number of Helicobacter pylori CagA variable-region EPIYA tyrosine phosphorylation motifs by PCR

Helicobacter pylori strains possessing the cag pathogenicity island are associated with the development of gastric cancer. The CagA protein is translocated into epithelial cells and becomes phosphorylated on tyrosine residues within EPIYA motifs, which may be repeated within the variable region of the protein. Strains possessing CagA with greater numbers of these repeats have been more closely associated with gastric carcinogenesis. Phosphorylated CagA leads to epithelial cell elongation, which is dependent on the number of variable-region EPIYA motifs. Thus, determination of the degree of CagA phosphorylation and the number of EPIYA motifs appears to be more important than detection of cagA alone. Determination of the number of EPIYA motifs by nucleotide sequencing, however, is a laborious and expensive process. We describe here a novel and rapid PCR method for determination of the pattern of repeats containing the EPIYA motif. This will aid in the identification of those strains that may be more likely to cause disease.     

A Modular Framework for Development and Interlaboratory Sharing and Validation of Diffusion Tensor Tractography Algorithms

This Technical Note describes a novel modular framework for development and interlaboratory distribution and validation of 3D tractography algorithms based on in vivo diffusion tensor imaging (DTI) measurements. The proposed framework allows individual MRI research centers to benefit from new tractography algorithms developed at other independent centers by "plugging" new tractography modules directly into their own custom DTI software tools, such as existing graphical user interfaces (GUI) for visualizing brain white matter pathways. The proposed framework is based on the Java 3D programming platform, which provides an object-oriented programming (OOP) model and independence of computer hardware configuration and operating system. To demonstrate the utility of the proposed approach, a complete GUI for interactive DTI tractography was developed, along with two separate and interchangeable modules that implement two different tractography algorithms. Although the application discussed here relates to DTI tractography, the programming concepts presented here should be of interest to anyone who wishes to develop platform-independent GUI applications for interactive 3D visualization.     

Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins.

Adherence of Candida albicans to host tissues is a necessary step for maintenance of its commensal status and is likely a necessary step in the pathogenesis of candidiasis. The extracellular matrix (ECM) proteins are some of the host tissue and plasma proteins to which C. albicans adheres through adhesins located on the fungal cell surface. To isolate genes encoding ECM adhesins, an assay was developed based on the ability of yeast cells to adhere to magnetic beads coated with the ECM protein fibronectin, type IV collagen, or laminin. A C. albicans genomic library was constructed by cloning XbaI-partially-digested and size-selected fragments into pAUR112, an Escherichia coli-yeast low-copy-number shuttle vector. The C. albicans library was transformed into Saccharomyces cerevisiae YPH 499, and clones capable of adherence were selected by using ECM protein-coated magnetic beads. A plasmid containing an approximately 8-kb insert was isolated from 29 adherent clones. These clones exhibited adherence to all ECM protein-coated magnetic beads and to human buccal epithelial cells. The ALA1 gene (for agglutinin-like adhesin) was localized by subcloning it into a 5-kb XbaI fragment which retained the adherence phenotype in both orientations. The complete DNA sequence of the 5-kb insert was determined, and an open reading frame (ORF) encoding 1,419 amino acid residues was identified. Deletions from the 5' and 3' ends extending into the DNA sequence encoding the 1,419-amino-acid ORF product inactivated the adherence phenotype, suggesting that it is the coding region of the ALA1 gene. A database search identified ALA1 to be similar to the C. albicans ALS1 (for agglutinin-like sequence 1) protein and the S. cerevisiae agglutinin protein (AG alpha1), although the homology at the primary amino acid sequence level is limited to the first half of each of these proteins. ALA1 contains a central domain of six tandem repeats of 36 amino acids. We discuss the significance of various predicted ALA1 structural motifs and their relationships to function in the adherence process.     

ViewGene: a graphical tool for polymorphism visualization and characterization

The human genome project is producing an enormous amount of sequence data, based on which single base changes between individuals can be identified. Unfortunately, computer tools that were adequate for sequence assembly are less than ideal for the characterization of polymorphism data [single nucleotide (snp) or insertion/deletion (indel)] and other sequence features, and their relationship to each other. We have developed viewGene as a flexible tool that takes input from a number of sequence formats and analysis programs (Genbank, FASTA, RepeatMasker, Cross match, BLAST, user-defined data) to construct a sequence reference scaffold that can be viewed through a simple graphical interface. polymorphisms generated from many sources can be added to this scaffold through the same sequence formats, with a variety of options to control what is displayed. Large amounts of polymorphism data can be organized so that patterns and haplotypes can be readily discerned. In our laboratory, viewGene has been used to view annotated genbank records, find nonrepetitive sequence fragments for polymorphism detection, and visualize similarity search results. Manipulation, cross-referencing, and haplotype viewing of snp data are essential for quality assessment and identification of variants associated with genetic disease, and viewGene provides all three of these important functions.     

Immunohistologic evaluation of metastatic carcinomas of unknown origin: an algorithmic approach

Pathologists are now asked frequently to determine the primary site for metastatic carcinomas of unknown origin (MCUO), using adjunctive morphological techniques such as electron microscopy, immunohistology, and other modalities. The authors present an algorithmic immunohistochemical approach to this problem that is based on their experience with over 2,800 routinely-processed epithelial malignancies of various types. These have been studied with antibodies to keratins, vimentin, epithelial membrane antigen, MOC-31, tumor associated-glycoprotein-72 (recognized by monoclonal antibody B72.3), prostate-specific antigen, thyroglobulin, gross cystic disease fluid protein-15, carcinoembryonic antigen, CA-125, CA19-9, placental alkaline phosphatase, S100 protein, and estrogen receptor protein. The algorithm that is structured around these 14 analytes is based on the relative predictive value of each marker, which in turn, determines its place in the sequence of interpretation. The authors' experience with this approach shows 67% accuracy with regard to the ultimately determined site of origin for MCUO, a figure which is similar to that reported by other investigators.     

A Role for Non‐B DNA Forming Sequences in Mediating Microlesions Causing Human Inherited Disease

Missense/nonsense mutations and microdeletions/microinsertions (<21 bp) represent ～76% of all mutations causing human inherited disease, and their occurrence has been associated with sequence motifs (direct, inverted, and mirror repeats; G‐quartets) capable of adopting non‐B DNA structures. We found that a significant proportion (～21%) of both microdeletions and microinsertions occur within direct repeats, and are explicable by slipped misalignment. A novel mutational mechanism, DNA triplex formation followed by DNA repair, may explain ～5% of microdeletions and microinsertions at mirror repeats. Further, G‐quartets, direct, and inverted repeats also appear to play a prominent role in mediating missense mutations, whereas only direct and inverted repeats mediate nonsense mutations. We suggest a mutational mechanism involving slipped strand mispairing, slipped structure formation, and DNA repair, to explain ～15% of missense and ～12% of nonsense mutations yielding perfect direct repeats from imperfect repeats, or the extension of existing direct repeats. Similar proportions of missense and nonsense mutations were explicable by hairpin/loop formation and DNA repair, yielding perfect inverted repeats from imperfect repeats. We also propose a model for single base‐pair substitution based on one‐electron oxidation reactions at G‐quadruplex DNA. Overall, the proposed mechanisms provide support for a role for non‐B DNA structures in human gene mutagenesis.     

Vacuolar targeting of aldehyde dehydrogenase 6 tagging with signal peptide of proteinase A

Vacuoles are useful materials with antimicrobial and anticancerous properties. Vacuolar proteins can discompose macromolecules from the outside of yeast cells. The objective of this study was to determine the function of a protein transported into a vacuole. Specifically, cytosolic protein aldehyde dehydrogenase 6 (ALD6) was used for the delivery to the vacuole. To transport cytosolic protein to the vacuole in this study, a transfer vector including a signal peptide sequence isolated from vacuolar protein proteinase A was designed. A signal peptide is an amino acid sequence in front of the transported protein. Signal peptides have various delivery pathways according to the kind of signal sequence they contain. They play important roles in transporting proteins to organelles, in cellular mechanisms, and the transfer of protein outside and inside eukaryotes. Thus, we focused on the design of a transfer vector containing a signal peptide sequence isolated from the DNA sequence of proteinase A (PEP4). In addition, this study evaluated the expression level of cytosolic ALD6 after being transported into the yeast vacuole. Our results showed that the developed transfer vector was useful for delivering proteins to vacuole by using signal peptide sequence. Therefore, this transfer vector might be used as a tool to deliver target proteins to organelles of interest in eukaryotes.     

Comparative analysis of the polycystic kidney disease 1 (PKD1) gene reveals an integral membrane glycoprotein with multiple evolutionary conserved domains

PKD1 is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease (ADPKD). Analysis of the predicted protein sequence of the human PKD1 gene, polycystin, shows a large molecule with a unique arrangement of extracellular domains and multiple putative transmembrane regions. The precise function of polycystin remains unclear with a paucity of mutations to define key structural and functional domains. To refine the structure of this protein we have cloned the genomic region encoding the Fugu PKD1 gene. Fugu PKD1 spans 36 kb of genomic DNA and has greater complexity with 54 exons compared with 46 in man. Comparative analysis of the predicted protein sequences shows a lower level of homology than in similar studies with identity of 40 and 59% similarity. However key structural motifs including leucine rich repeats (LRR), a C-type lectin and LDL-A like domains and 16 PKD repeats are maintained. A region of homology with the sea urchin REJ protein was also confirmed in Fugu but found to extend over 1000 amino acids. Several highly conserved intra- and extra- cellular regions, with no known sequence homologies, that are likely to be of functional importance were detected. The likely structure of the membrane associated region has been refined with similarity to the PKD2 protein and voltage gated Ca2+ and Na+ channels highlighted over part of this area. The overall protein structure has therefore been clarified and this comparative analysis derived structure will form the basis for the functional study of polycystin and its individual domains.