bluuebunny/crossref_metadata_2025 huggingface dataset:
Overlapping PCR for Bidirectional PCR Amplification of Specific Alleles: A Rapid One-Tube Method for Simultaneously Differentiating Homozygotes and Heterozygotes
Authors: Liu Qiang , Thorland Erik C. , Heit John A. , Sommer Steve S.
Published: May 2017

Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification ofspecific alleles) is a rapid method of genotyping single-base changes, but one reaction is required for each allele. Bidirectional PASA (Bi-PASA) was developed to distinguish between homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. In Bi-PASA, one of the alleles is amplified by a PASA reaction in one direction while the second allele is amplified by a PASA reaction in the opposite direction. Two outer (P and Q) and two inner allele-specific (A and B) primers are required. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify. The two inner primers (A and B) contain a relatively short complementary region and a 10-nucleotide G+C-rich 5′ tail. The inner primers “switch” from low-efficiency to high-efficiency amplification when genomic DNA is replaced by previously amplified template DNA. In addition, the 5′ tails prevent “megapriming”. The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human factor V and catechol–O-methyltransferase genes. Guidelines for optimization of Bi-PASA also were developed and tested in a prospective study. Three additional Bi-PASA assays were optimized rapidly by utilizing these guidelines. In conclusion, Bi-PASA is a simple and rapid method for detecting the zygosity of known mutations in a single PCR reaction.
Clone–Contig and STS Maps of the Hereditary Hemochromatosis Region on Human Chromosome 6p21.3–p22
Authors: Lauer Peter , Meyer Nicole C. , Prass Cynthia E. , Starnes Steven M. , Wolff Roger K. , Gnirke Andreas
Published: May 2017

YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis. The YAC-based map comprises 43 YACs and 86 STS and spans ∼8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22. Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself. Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 P1 clones and one λ phage. The bacterial clone-based STS map comprises 153 STSs. A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5. Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5. Possible implications of the incomplete public YAC–contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed.

[The sequence data described in this paper have been submitted to GenBank under accession nos. G31205–G31325.]
Low Frequency of Microsatellites in the Avian Genome
Authors: Primmer Craig R. , Raudsepp Terje , Chowdhary Bhanu P. , Møller Anders Pape , Ellegren Hans
Published: May 2017

A better insight into the occurrence of microsatellites in a range of taxa may help to understand the evolution of simple repeats. Previous studies have found the relative abundance of several repeat motifs to differ among mammals, invertebrates, and plants. Absolute numbers of microsatellites also tend to correlate positively with genome size. We analyzed the occurrence, frequency, and distribution of microsatellites in birds, a taxon with one of the smallest known genome sizes among vertebrates. Dot-blot hybridization revealed that about half of 22 different di-, tri-, and tetranucleotide repeat motifs were clearly more common in human than in three species of birds: chicken, woodpecker, and swallow. For the remaining motifs no clear difference was found. From searching avian database sequences we estimated there to be 30,000–70,000 microsatellites longer than 20 bp in the avian genome. The number of (CA)⩾10 would be around 7000–9000 and the number of (CA)⩾14 about 3000. The calculated density of avian microsatellites (total, one every 20–39 kb; (CA)⩾10, one every 136–150 kb) is much lower than that estimated for the human genome (one every 6 and 30 kb, respectively). This may be explained by the fact that the avian genome contains relatively less noncoding DNA than most mammals and that avian SINE/LINE elements do not terminate in poly(A) tails, which are known to provide a resource for the evolution of simple repeats in mammals. We found no association between microsatellites and SINEs in birds. Primed in situ labeling suggested fairly even distribution of (CA)n repeats over chicken macrochromosomes and intermediate chromosomes, whereas the microchromosomes, a large part of the Z and W chromosomes, and most telomeres and centromeres had very low concentrations of (CA)n microsatellites. The scarcity of microsatellites on the microchromosomes is compatible to these regions likely being unusually rich in coding sequences. The low microsatellite density in the genome in general and on the microchromosomes in particular imposes an obstacle for the development of marker-rich genetic maps of chicken and other birds, and for the localization of quantitative trait genes.
Fine Localization of the Torsion Dystonia Gene (DYT1) on Human Chromosome 9q34: YAC Map and Linkage Disequilibrium
Authors: Ozelius Laurie J. , Hewett Jeffrey , Kramer Patricia , Bressman Susan B. , Shalish Christo , de Leon Deborah , Rutter Marc , Risch Neil , Brin Mitchell F. , Markova Elena D. , Limborska Svetlana A. , Ivanova-Smolenskaya Irina A. , McCormick Mary Kay , Fahn Stanley , Buckler Alan J. , Gusella James F. , Breakefield Xandra O.
Published: May 2017

The DYT1 gene, which maps to chromosome 9q34, appears to be responsible for most cases of early-onset torsion dystonia in both Ashkenazic Jewish (AJ) and non-Jewish families. This disease is inherited in an autosomal dominant mode with reduced penetrance (30%–40%). The abnormal involuntary movements associated with this disease are believed to be caused by unbalanced neural transmission in the basal ganglia. Previous linkage disequilibrium studies in the AJ population placed the DYT1 gene in a 2-cM region between the loci D9S62a and ASS. A YAC contig has now been created spanning 600 kb of this region including D9S62a. The location of the DYT1 gene has been refined within this contig using several new polymorphic loci to expand the linkage disequilibrium analysis of the AJ founder mutation. The most likely location of theDYT1 gene is within a 150 kb region between the lociD9S2161 and D9S63.
Computational and Biological Analysis of 680 kb of DNA Sequence from the Human 5q31 Cytokine Gene Cluster Region
Authors: Frazer Kelly A. , Ueda Yukihiko , Zhu Yiwen , Gifford Vincent R. , Garofalo Maria R. , Mohandas Narla , Martin Christopher H. , Palazzolo Michael J. , Cheng Jan-Fang , Rubin Edward M.
Published: May 2017

With the human genome project advancing into what will be a 7- to 10-year DNA sequencing phase, we are presented with the challenge of developing strategies to convert genomic sequence data, as they become available, into biologically meaningful information. We have analyzed 680 kb of noncontiguous DNA sequence from a 1-Mb region of human chromosome 5q31, coupling computational analysis with gene expression studies of tissues isolated from humans as well as from mice containing human YAC transgenes. This genomic interval has been noted previously for containing the cytokine gene cluster and a quantitative trait locus associated with inflammatory diseases. Our analysis identified and verified expression of 16 new genes, as well as 7 previously known genes. Of the total of 23 genes in this region, 78% had similarity matches to sequences in protein databases and 83% had exact expressed sequence tag (EST) database matches. Comparative mapping studies of eight of the new human genes discovered in the 5q31 region revealed that all are located in the syntenic region of mouse chromosome 11q. Our analysis demonstrates an approach for examining human sequence as it is made available from large sequencing programs and has resulted in the discovery of several biomedically important genes, including a cyclin, a transcription factor that is homologous to an oncogene, a protein involved in DNA repair, and several new members of a family of transporter proteins.

[The sequence data described in this paper are available via the internet athttp://www-hgc.lbl.gov/sequencearchive.html.]
Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays
Authors: Pastinen Tomi , Kurg Ants , Metspalu Andres , Peltonen Leena , Syvänen Ann-Christine
Published: May 2017

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.
Cloning of Two Human Homologs of the Drosophila single-minded Gene SIM1 on Chromosome 6q and SIM2 on 21q Within the Down Syndrome Chromosomal Region
Authors: Chrast Roman , Scott Hamish S. , Chen Haiming , Kudoh Jun , Rossier Colette , Minoshima Shinsei , Wang Yimin , Shimizu Nobuyoshi , Antonarakis Stylianos E.
Published: May 2017

As part of our effort to clone genes of human chromosome 21 that may contribute to Down syndrome, we have previously isolated four exons with homology to Drosophila single-minded (sim) gene, which encodes a transcription factor that is a master regulator of fruit fly neurogenesis. These exons were used to clone and characterize two human homologs of the Drosophila sim gene, SIM1 and SIM2, which map to chromosomes 6q16.3–q21 and 21q22.2, respectively;SIM2 maps within the so-called Down syndrome chromosomal region. Recently, two mouse homologs, Sim1 and Sim2, also have been identified. There is a high level of homology among human, mouse, andDrosophila sim genes in their amino-terminal half where the conserved bHLH, PAS1, PAS2, and HST domains are present. In contrast, the carboxy-terminal parts are only homologous between SIM1 and Sim1 and SIM2 and Sim2. Two isoforms (SIM2 and SIM2s) of human SIM2 have been detected that differ in their 3′ ends. Northern blot analysis revealed one mRNA SIM1 species of ∼9.5 kb and four different mRNA SIM2 species of 2.7, 3, 4.4, and 6 kb in human fetal kidney. The function of both human SIM1 and SIM2 is unknown. However, three copies of SIM2 may contribute to some specific Down syndrome phenotypes because of (1) mapping position, (2) potential function as transcriptional repressor, (3) likely dimerization with other transcription factors, (4) the temporal and spatial expression pattern of mouse Sim2, and (5) the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis.

[The sequence data described in this paper have been submitted to GenBank under accession nos. U70212, U80456, U80457, and AB003185.]
PowerBLAST: A New Network BLAST Application for Interactive or Automated Sequence Analysis and Annotation
Authors: Zhang Jinghui , Madden Thomas L.
Published: May 2017

As the rate of DNA sequencing increases, analysis by sequence similarity search will need to become much more efficient in terms of sensitivity, specificity, automation potential, and consistency in annotation. PowerBLAST was developed, in part, to address these problems. PowerBLAST includes a number of options for masking repetitive elements and low complexity subsequences. It also has the capacity to restrict the search to any level of NCBI’s taxonomy index, thus supporting “comparative genomics” applications. Postprocessing of the BLAST output using the SIM series of algorithms produces optimal, gapped alignments, and multiple alignments when a region of the query sequence matches multiple database sequences. PowerBLAST is capable of processing sequences of any length because it divides long query sequences into overlapping fragments and then merges the results after searching. The results may be viewed graphically, as a textual representation, or as an HTML page with links to GenBank and Entrez. For matching database sequences, annotated features are superimposed on the aligned query sequence in the output, thus greatly increasing the ease of interpretation. Such features may be used for automated annotation of new sequence because PowerBLAST output in ASN.1 form may be “dragged and dropped” into NCBI’s Sequin program for sequence annotation and submission. PowerBLAST is capable of analyzing and annotating a 100-kb query in 60 min on NCBI’s BLAST server.

[THC BLAST is available athttp://www.ncbi.nlm.nih.gov/cgi-bin/THCBlast/nph-thcblast]
Resistance to Salmonellosis in the Chicken Is Linked toNRAMP1and TNC
Authors: Hu Jinxin , Bumstead Nat , Barrow Paul , Sebastiani Giovanna , Olien Leonard , Morgan Kenneth , Malo Danielle
Published: May 2017

Natural resistance to infection withSalmonella typhimuriumin mice is controlled by two major loci,BcgandLps,located on mouse chromosomes 1 and 4, respectively. BothBcgandLpsexert pleiotropic effects and contribute to cytostatic/cytocidal activities of the macrophage.Bcgencodes for a membrane phosphoglycoprotein designatedNramp1(natural resistance-associated macrophage protein 1), which belongs to an ancient family of membrane proteins.Lpshas not been cloned yet, but its location on mouse chromosome 4 has been refined for positional cloning. As in mice, chicken inbred lines differ in their susceptibility to infection withSalmonella typhimurium.We have tested the candidacy of the chicken homologs ofNramp1andTnc(a locus closely linked toLps), in the differential resistance of chicken inbred lines to infection withS. typhimurium.We have first analyzed six inbred chicken lines ofSalmonella-resistant orSalmonella-susceptible phenotypes for the presence of nucleotide sequence variations within the coding portion ofNRAMP1.We have identified 11 sequence variations withinNRAMP1in the chicken inbred lines tested: 10 of these represented either silent mutations or conservative changes. However, one G → A substitution at nucleotide 696 resulted in the nonconservative replacement of Arg223to Gln223within the predicted TM5-6 region. This allelic variant was specific to the susceptible line C and not observed in any of the resistant strains. To investigate the effect ofNRAMP1andTNCon resistance to infection withS. typhimurium,425 (W1 × C)F1 × C chicken progeny were examined during a period of 15 days postinfection. Together,NRAMP1andTNCexplain 33% of the early differential resistance to infection withS. typhimuriumof parental lines C and W1. Our data established that resistance to infection withS. typhimuriumin chickens is inherited as a complex trait and that comparative mapping has proven to be useful to identifySalmonella-resistance genes in the chicken.
High-Resolution Physical and Transcriptional Mapping of the Autoimmune Polyendocrinopathy–Candidiasis–Ectodermal Dystrophy Locus on Chromosome 21q22.3 by FISH
Authors: Aaltonen Johanna , Horelli-Kuitunen Nina , Fan Jian-Bing , Björses Petra , Perheentupa Jaakko , Myers Richard , Palotie Aarno , Peltonen Leena
Published: May 2017

Autoimmune–polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED, PGD type I) is an autosomal recessive disease enriched in the Finnish population. Previously, we have assigned APECED to a 2.6-cM interval on chromosome 21q22.3 by linkage analysis in 14 Finnish families. This subtelomeric region of 21q22.3 seems to have sequence features resulting in its under-representation in large insert genomic libraries, and only a few large insert clones have been available for positional cloning to date. Here, we report the refined localization of the APECED gene and a visual physical map of 800 kb covering the critical chromosomal region for the gene. In the construction of the physical map, the recently developed fiber FISH techniques were essential for the orientation of the cosmid P1, PAC, and BAC clones in relation to each other. We also localized two cDNAs within this genomic region by fiber FISH combined with the highly sensitive tyramide-based detection method. These data will facilitate the final cloning of theAPECED gene and any other novel gene in this complex genomic region.

[On-line supplement for primer sequences, PCR product size, and annealing temperatures is available athttp://www.cshl.org/gr.]
Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence
Authors: André Paulo , Kim Andrea , Khrapko Konstantin , Thilly William G.
Published: May 2017

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived fromPyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) ofPfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.
A Radiation Hybrid Map of Human Chromosome 5 with Integration of Cytogenetic, Genetic, and Transcript Maps
Authors: McPherson John D. , Apostol Barbara , Wagner-McPherson Caryn B. , Hakim Simin , Del Mastro Richard G. , Aziz Naeema , Baer Elizabeth , Gonzales Genalyn , Krane Mary Carol , Markovich Rachelle , Masny Peter , Ortega Miguel , Vu John , Vujicic Marco , Church Deanna M. , Segal Allan , Grady Deborah L. , Moyzis Robert K. , Spence M. Anne , Lovett Michael , Wasmuth John J.
Published: May 2017

One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.

[The STS sequences described in this paper have been submitted to dbSTS under accession nos. G15666–G15715 andG16049–G16063. A complete map of human chromosome 5 is available as an on-line supplement at http://www.cshl.org/gr.]
CpG Islands of the Pig
Authors: McQueen Heather A. , Clark Victoria H. , Bird Adrian P. , Yerle Martine , Archibald Alan L.
Published: May 2017

We describe an analysis of the CpG islands (CGIs) of the pig. We have used both database survey and a porcine genomic library that is enriched for CGIs. Approximately half of 41 pig genomic database sequences had CGIs with an average G + C content of 65.3%, an average CpG observed/expected frequency of 0.85, and an average size of 978 bp. Of 27 CGI library clones, 16 were nonrepetitive, nonribosomal DNA and CGI-like. CGI library clones had similar average values for G + C and CpG frequency to CGIs of database genes, and an average size of 670 bp, as MseI cuts within some islands. Library clones were also shown to be low copy number and unmethylated in genomic DNA. The presence in the library of seven previously known CGI sequences was confirmed as was the absence of one nonisland sequence. The CGI library exhibits an R-band pattern for many chromosomes in FISH analysis. The pig chromosome arms that show the most dense CGI population are homologous to segments of human chromosomes that are known to be gene rich.

[The sequence data described in this paper have been submitted to the EMBL data library under accession nos.Y10585–Y10592, Y10594, Y10596–Y10597.]
High-Precision Genotyping by Denaturing Capillary Electrophoresis
Authors: Wenz H.-Michael , Robertson James M. , Menchen Steve , Oaks Frank , Demorest David M. , Scheibler Don , Rosenblum Barnett B. , Wike Carla , Gilbert Dennis A. , Efcavitch J. William
Published: May 2017

Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of ±0.3 nucleotide are required to ensure with 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, operation at 60°C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d’Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.
Pathological Consequences of Sequence Duplications in the Human Genome
Authors: Mazzarella Richard , Schlessinger David
Published: May 2017

As large-scale sequencing accumulates momentum, an increasing number of instances are being revealed in which genes or other relatively rare sequences are duplicated, either in tandem or at nearby locations. Such duplications are a source of considerable polymorphism in populations, and also increase the evolutionary possibilities for the coregulation of juxtaposed sequences. As a further consequence, they promote inversions and deletions that are responsible for significant inherited pathology. Here we review known examples of genomic duplications present on the human X chromosome and autosomes.
Complete Genomic Sequence and Analysis of the Prion Protein Gene Region from Three Mammalian Species
Authors: Lee Inyoul Y. , Westaway David , Smit Arian F.A. , Wang Kai , Seto Jason , Chen Lei , Acharya Chetana , Ankener Mike , Baskin Dale , Cooper Carol , Yao Hong , Prusiner Stanley B. , Hood Leroy E.
Published: May 2017

The prion protein (PrP), first identified in scrapie-infected rodents, is encoded by a single exon of a single-copy chromosomal gene. In addition to the protein-coding exon, PrP genes in mammals contain one or two 5′-noncoding exons. To learn more about the genomic organization of regions surrounding the PrP exons, we sequenced 105 bp of DNA from clones containing human, sheep, and mouse PrP genes isolated in cosmids or λ phage. Our findings are as follows: (1) Although the human PrP transcript does not include the untranslated exon 2 found in its mouse and sheep counterparts, the large intron of the human PrP gene contains an exon 2-like sequence flanked by consensus splice acceptor and donor sites. (2) The mouse Prnpa but not thePrnpb allele found in 44 inbred lines contains a 6593 nucleotide retroviral genome inserted into the anticoding strand of intron 2. This intracisternal A-particle element is flanked by duplications of an AAGGCT nucleotide motif. (3) We found that thePrP gene regions contain from 40% to 57% genome-wide repetitive elements that independently increased the size of the locus in all three species by numerous mutations. The unusually long sheepPrP 3′-untranslated region contains a “fossil” 1.2-kb mariner transposable element. (4) We identified sequences in noncoding DNA that are conserved between the three species and may represent biologically functional sites.

[The nucleotide sequence data reported in this paper have been submitted to the GenBank sequence database and have been assigned the accession numbers U29185(human), U29186 (mouse), and U67922 (sheep).]
Large-Scale Sequencing of Two Regions in Human Chromosome 7q22: Analysis of 650 kb of Genomic Sequence around the EPO and CUTL1 Loci Reveals 17 Genes
Authors: Glöckner Gernot , Scherer Stephen , Schattevoy Ruben , Boright Andrew , Weber Jacqueline , Tsui Lap-Chee , Rosenthal André
Published: May 2017

We have sequenced and annotated two genomic regions located in the Giemsa negative band q22 of human chromosome 7. The first region defined by the erythropoietin (EPO) locus is 228 kb in length and contains 13 genes. Whereas 3 genes (GNB2, EPO,PCOLCE) were known previously on the mRNA level, we have been able to identify 10 novel genes using a newly developed automatic annotation tool RUMMAGE-DP, which comprises >26 different programs mainly for exon prediction, homology searches, and compositional and repeat analysis. For precise annotation we have also resequenced ESTs identified to the region and assembled them to build large cDNAs. In addition, we have investigated the differential splicing of genes. Using these tools we annotated 4 of the 10 genes as a zonadhesin, a transferrin homolog, a nucleoporin-like gene, and an actin gene. Two genes showed weak similarity to an insulin-like receptor and a neuronal protein with a leucine-rich amino-terminal domain. Four predicted genes (CDS1–CDS4) CDS that have been confirmed on the mRNA level showed no similarity to known proteins and a potential function could not be assigned. The second region in 7q22 defined by the CUTL1 (CCAAT displacement protein and its splice variant) locus is 416 kb in length and contains three known genes, includingPMSL12, APS, CUTL1, and a novel gene (CDS5). The CUTL1 locus, consisting of two splice variants (CDP and CASP), occupies >300 kb. Based on the G,C profile an isochore switch can be defined between theCUTL1 gene and the APS and PMSL12genes.

[Clones 37G3, 164c7, and 235f8 are deposited in GenBank under accession no. AF053356; clone 123e15, accession no.AF024533; 186d2, accession no. AF024534; 46f6, accession no. AF006752; 50h2, accession no. AF047825; and 76h2, accession no. AF030453]
Analysis of the Quality and Utility of Random Shotgun Sequencing at Low Redundancies
Authors: Bouck John , Miller Webb , Gorrell James H. , Muzny Donna , Gibbs Richard A.
Published: May 2017

The currently favored approach for sequencing the human genome involves selecting representative large-insert clones (100–200 kb), randomly shearing this DNA to construct shotgun libraries, and then sequencing many different isolates from the library. This method, entitled directed random shotgun sequencing, requires highly redundant sequencing to obtain a complete and accurate finished consensus sequence. Recently it has been suggested that a rapidly generated lower redundancy sequence might be of use to the scientific community. Low-redundancy sequencing has been examined previously using simulated data sets. Here we utilize trace data from a number of projects submitted to GenBank to perform reconstruction experiments that mimic low-redundancy sequencing. These low-redundancy sequences have been examined for the completeness and quality of the consensus product, information content, and usefulness for interspecies comparisons. The data presented here suggest three different sequencing strategies, each with different utilities. (1) Nearly complete sequence data can be obtained by sequencing a random shotgun library at sixfold redundancy. This may therefore represent a good point to switch from a random to directed approach. (2) Sequencing can be performed with as little as twofold redundancy to find most of the information about exons, EST hits, and putative exon similarity matches. (3) To obtain contiguity of coding regions, sequencing at three- to fourfold redundancy would be appropriate. From these results, we suggest that a useful intermediate product for genome sequencing might be obtained by three- to fourfold redundancy. Such a product would allow a large amount of biologically useful data to be extracted while postponing the majority of work involved in producing a high quality consensus sequence.
Clusters of Resistance Genes in Plants Evolve by Divergent Selection and a Birth-and-Death Process
Authors: Michelmore Richard W. , Meyers Blake C.
Published: May 2017

Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (Rgenes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate thatRgenes are part of signal-transduction systems. Most of theseRgenes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individualRgenes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.
Snapshot of a Large Dynamic Replicon in a Halophilic Archaeon: Megaplasmid or Minichromosome?
Authors: Ng WaiLap V. , Ciufo Stacy A. , Smith Todd M. , Bumgarner Roger E. , Baskin Dale , Faust Janet , Hall Barbara , Loretz Carol , Seto Jason , Slagel Joseph , Hood Leroy , DasSarma Shiladitya
Published: May 2017

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, inHalobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

[The sequence data described in this paper have been submitted to GenBank under accession no. AF016485.]
How to Interpret an Anonymous Bacterial Genome: Machine Learning Approach to Gene Identification
Authors: Hayes William S. , Borodovsky Mark
Published: May 2017

In this report we address the problem of accurate statistical modeling of DNA sequences, either coding or noncoding, for a bacterial species whose genome (or a large portion) was sequenced but not yet characterized experimentally. Availability of these models is critical for successful solution of the genome annotation task by statistical methods of gene finding. We present the method, GeneMark-Genesis, which learns the parameters of Markov models of protein-coding and noncoding regions from anonymous bacterial genomic sequence. These models are subsequently used in the GeneMark and GeneMark.hmm gene-finding programs. Although there is basically one model of a noncoding region for a given genome, several models of protein-coding region are automatically obtained by GeneMark-Genesis. The diversity of protein-coding models reflects the diversity of oligonucleotide compositions, particularly the diversity of codon usage strategies observed in genes from one and the same genome. In the simplest and the most important case, there are just two gene models—typical and atypical ones. We show that the atypical model allows one to predict genes that escape identification by the typical model. Many genes predicted by the atypical model appear to be horizontally transferred genes. The early versions of GeneMark-Genesis were used for annotating the genomes of Methanoccocus jannaschii and Helicobacter pylori. We report the results of accuracy testing of the full-scale version of GeneMark-Genesis on 10 completely sequenced bacterial genomes. Interestingly, the GeneMark.hmm program that employed the typical and atypical models defined by GeneMark-Genesis was able to predict 683 new atypical genes with 176 of them confirmed by similarity search.
Predicting Gene Regulatory Elements in Silico on a Genomic Scale
Authors: Brāzma Alvis , Jonassen Inge , Vilo Jaak , Ukkonen Esko
Published: May 2017

We performed a systematic analysis of gene upstream regions in the yeast genome for occurrences of regular expression-type patterns with the goal of identifying potential regulatory elements. To achieve this goal, we have developed a new sequence pattern discovery algorithm that searches exhaustively for a priori unknown regular expression-type patterns that are over-represented in a given set of sequences. We applied the algorithm in two cases, (1) discovery of patterns in the complete set of >6000 sequences taken upstream of the putative yeast genes and (2) discovery of patterns in the regions upstream of the genes with similar expression profiles. In the first case, we looked for patterns that occur more frequently in the gene upstream regions than in the genome overall. In the second case, first we clustered the upstream regions of all the genes by similarity of their expression profiles on the basis of publicly available gene expression data and then looked for sequence patterns that are over-represented in each cluster. In both cases we considered each pattern that occurred at least in some minimum number of sequences, and rated them on the basis of their over-representation. Among the highest rating patterns, most have matches to substrings in known yeast transcription factor-binding sites. Moreover, several of them are known to be relevant to the expression of the genes from the respective clusters. Experiments on simulated data show that the majority of the discovered patterns are not expected to occur by chance.
Strategies for Mutational Analysis of the Large Multiexon ATM Gene Using High-Density Oligonucleotide Arrays
Authors: Hacia Joseph G. , Sun Bryan , Hunt Nathaniel , Edgemon Keith , Mosbrook Deborah , Robbins Christiane , Fodor Stephen P.A. , Tagle Danilo A. , Collins Francis S.
Published: May 2017

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATMcoding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.
Scriptable Access to the Caenorhabditis elegans Genome Sequence and Other ACEDB Databases
Authors: Stein Lincoln D. , Thierry-Mieg Jean
Published: May 2017

Much of the world’s genomic data are available to the community through networked databases that are accessed via Web interfaces. Although this paradigm provides browse-level access and has greatly facilitated linking between databases, it does not provide any convenient mechanism for programmatically fetching and integrating data from diverse databases. We have created a library and an application programming interface (API) named AcePerl that provides simple, direct access to ACEDB databases from the Perl programming language. With this library, programmers and computer-savvy biologists can write software to pose complex queries on local and remote ACEDB databases, retrieve the data, integrate the results, and move data objects from one database to another. In addition, a set of Web scripts running on top of AcePerl provides Web-based browsing of any local or remote ACEDB database. AcePerl and the AceBrowser Web browser run on Unix systems and are available under a license that allows for unrestricted use and redistribution. Both packages can be downloaded from URLhttp://stein.cshl.org/AcePerl. A Microsoft Windows port of AcePerl is in the planning stages.
The Distribution of Variation in Regulatory Gene Segments, as Present in MHC Class II Promoters
Authors: Cowell Lindsay G. , Kepler Thomas B. , Janitz Michael , Lauster Roland , Mitchison N. Avrion
Published: May 2017

Diversity in the antigen-binding receptors of the immune system has long been a primary interest of biologists. Recently it has been suggested that polymorphism in regulatory (noncoding) gene segments is of substantial importance as well. Here, we survey the level of variation in MHC class II gene promoters in man and mouse using extensive collections of published sequences together with unpublished sequences recently deposited by us in the EMBL gene bank using the Shannon entropy to quantify diversity. For comparison, we also apply our analysis to distantly related MHC class II promoters, as well as to class I promoters and to class II coding regions. We observe a high level of intraspecies variability, which in mouse but not in man is localized to a significant extent near the binding sites of transcription factors—sites that are conserved over longer evolutionary distances. This localization may both indicate and enhance heterozygote advantage, as the presence of two functionally different promoters would be expected to confer flexibility in the immune response.
A Physical Map of the Leishmania majorFriedlin Genome
Authors: Ivens Alasdair C. , Lewis Sharon M. , Bagherzadeh Azadeh , Zhang Linglan , Chan Ho M. , Smith Deborah F.
Published: May 2017

An extensive physical map of the Leishmania majorFriedlin genome has been assembled by the combination of fingerprint analysis of a shuttle vector cosmid library and probe hybridization. The integrated data obtained for 9004 fingerprinted clones and 974 probes have placed 91.2% of the 33.58-Mb genome into contigs representing each of the 36 chromosomes. This first-generation map has already provided a suitable framework for both high-throughput DNA sequencing and functional studies of the L. major parasite.
Integrated YAC Contig Map of the Prader–Willi/Angelman Region on Chromosome 15q11–q13 with Average STS Spacing of 35 kb
Authors: Christian Susan L. , Bhatt Nehal K. , Martin Scott A. , Sutcliffe James S. , Kubota Takeo , Huang Bing , Mutirangura Apiwat , Chinault A. Craig , Beaudet Arthur L. , Ledbetter David H.
Published: May 2017

Prader–Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11–q13, most frequently a deletion of an ∼4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5–149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11–q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11–q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.
New Insulin-Like Proteins with Atypical Disulfide Bond Pattern Characterized in Caenorhabditis elegans by Comparative Sequence Analysis and Homology Modeling
Authors: Duret Laurent , Guex Nicolas , Peitsch Manuel C. , Bairoch Amos
Published: May 2017

We have identified three new families of insulin homologs inCaenorhabditis elegans. In two of these families, concerted mutations suggest that an additional disulfide bond links B and A domains, and that the A-domain internal disulfide bond is substituted by a hydrophobic interaction. Homology modeling remarkably confirms these predictions and shows that despite this atypical disulfide bond pattern and the absence of C-like peptide, all these proteins may adopt the same fold as the insulin. Interestingly, whereas we identified 10 insulin-like peptides, only one insulin-like-receptor (daf-2) has been found. We propose that these insulin-related peptides may correspond to different activators or inhibitors of the daf-2insulin-regulating pathway.