bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative transcription map of the murine bare patches (Bpa) and striated (Str) critical regions and human Xq28.
Authors: Levin M L , Chatterjee A , Pragliola A , Worley K C , Wehnert M , Zhuchenko O , Smith R F , Lee C C , Herman G E
Published: June 2007

The X-linked developmental mouse mutations bare patches (Bpa) and striated (Str) may be homologous to human X-linked dominant chondrodysplasia punctata (CDPX2) and incontinentia pigmenti (IP2), respectively, based on their genetic mapping and clinical phenotypes. Bpa and Str have been localized to an overlapping critical region of 600 kb that demonstrates conserved gene order with loci in human Xq28 between DXS1104 and DXS52. As part of efforts to isolate the genes involved in these disorders, we have begun to develop a comparative transcription map spanning this region in both species. Using techniques of cross-species conservation and hybridization, exon trapping, and cDNA selection we have identified four known genes or members of gene families--caltractin, a member of the gamma-aminobutyric acid (GABAA) receptor gene family, a member of the melanoma antigen gene (MAGE) family, and several members of the murine-specific, X-linked lymphocyte regulated gene (Xlr3) family. Trapped exons and, in some cases, longer cDNAs have been isolated for potentially 7-9 additional genes. One cDNA demonstrates highly significant homology with members of the Krüppel family of zinc finger transcription factors. A second novel cDNA demonstrates homology at the 3' end of the predicted amino acid sequence to a LIM domain consensus. Gene order appears conserved among those cDNAs determined to be present in both human and mouse. Three of the murine transcripts appear to be present in multiple copies within the Bpa/Str critical region and could be associated with a predisposition to genomic rearrangements. Reverse transcriptase PCR (RT-PCR) and Northern analysis demonstrate that several of the transcripts are expressed in mid-gestation murine embryos and neonatal skin, making them candidates for the Bpa and Str mutations and their respective homologous human disorders.
Construction of a YAC contig encompassing the Usher syndrome type 1C and familial hyperinsulinism loci on chromosome 11p14-15.1.
Authors: Ayyagari R , Nestorowicz A , Li Y , Chandrasekharappa S , Chinault C , van Tuinen P , Smith R J , Hejtmancik J F , Permutt M A
Published: June 2007

The Usher syndrome type 1C (USH1C) and familial hyperinsulinism (HI) loci have been assigned to chromosome 11p14-15.1, within the interval D11S419-D11S1310. We have constructed a yeast artificial chromosome (YAC) contig, extending from D11S926 to D11S899, which encompasses the critical regions for both USH1C and HI and spans an estimated genetic distance of approximately 4 cM. A minimal set of six YAC clones constitute the contig, with another 22 YACs confirming the order of sequence-tagged sites (STSs) and position of YACs on the contig. A total of 40 STSs, including 10 new STSs generated from YAC insert-end sequences and inter-Alu PCR products, were used to order the clones within the contig. This physical map provides a resource for identification of gene transcripts associated with USH1C, HI, and other genetic disorders that map to the D11S926-D11S899 interval.
Octamer-primed cycle sequencing: design of an optimized primer library.
Authors: Hardin S H , Jones L B , Homayouni R , McCollum J C
Published: June 2007

This paper describes a novel method of primer walking using octamer oligonucleotides to prime DNA sequencing reactions. Octamer sequencing is compatible with isotopic and fluorescent sequencing chemistry, reaction conditions are optimized such that the samples can be processed in parallel, and the procedure has the potential to be automated. This strategy is faster than the traditional primer walking sequencing strategy, as the existence of a primer library allows immediate access to a primer for the next sequencing reaction, eliminating delays associated with designing and synthesizing gene-specific primers. The octamer library is comprised of optimized sequencing primers, such that octamer sequencing yields results equivalent to or better than traditional primer walking. This technology is more economical because gene-specific sequencing primers, the major cost in the reaction, are replaced by an optimized subset of frequently occurring octamers that are able to prime multiple reactions.
PCR induction of a TaqI restriction site at any CpG dinucleotide using two mismatched primers (CpG-PCR).
Authors: O'Dell S D , Humphries S E , Day I N
Published: June 2007

A final common pathway has been devised for analysis of de novo mutation at any CpG site. Artificial restriction sites can be introduced in known DNA sequences by using either or both sense and antisense mismatched PCR primers. Sitting of the primers directly adjacent 5' and 3' to the CpG site yields a 52-bp PCR product, resulting from the sum of the two 25-mer oligonucleotides plus the two intervening bases (C and G), and also yields consistent digestion fragments. Three out of four possible four-base palindromes (TaqI, HHaI, and MspI) were investigated for mutations R329X and E80K in the human LDL receptor gene, and for mutations R395W and R612C, and TaqI site was forced using PCR in which both primers had 3' mismatched T. Both empirically and on theoretical grounds, Taq1 is the forced restriction site of choice. The approach has been adapted to the high-throughput microplate diagonal gel electrophoresis (MADGE) system for selective analysis of mutations at CpG sites, which may account for 20% of all single base variation in the human genome.
Identity-by-descent mapping of recessive traits in livestock: application to map the bovine syndactyly locus to chromosome 15.
Authors: Charlier C , Farnir F , Berzi P , Vanmanshoven P , Brouwers B , Vromans H , Georges M
Published: June 2007

Twelve animals affected with syndactyly or mulefoot were sampled in the Dutch black-and-white cattle population. Analysis of the pedigree data reveal that all of these individuals traced back to a single acknowledged carrier founder individual. Between seven and nine generations separated the founder from its 12 affected descendents. The 12 affected offspring were genotyped for a battery of 213 microsatellites spanning the 29 bovine autosomes. The resulting genotypes were analyzed using a maximum likelihood approach searching for shared homozygous haplotypes among affected individuals. Three candidate regions for the syndactyly locus emerged from this initial screening. syndactyly was shown to map to one of these candidate regions on chromosome 15 by genotyping 29 additional individuals linking founder and affected offspring and performing a conventional linkage analysis with the LINKAGE programs. This study illustrates the potential of identity-by-descent mapping in livestock populations.
Worldwide distribution of human Y-chromosome haplotypes.
Authors: Santos F R , Bianchi N O , Pena S D
Published: June 2007

We surveyed several human populations worldwide with three PCR-based polymorphisms located in the human Y chromosome: the alphoid heteroduplex [alpha(h)] polymorphic system, the DYS19 microsatellite locus, and a polymorphic Alu insertion (YAP). By typing with the former two polymorphisms [alpha(h) and DYS19] we found 46 different haplotypes in 364 males from several populations worldwide. There were significant geographic differences in the distribution of the haplotypes, several of which were seen in only one population and can be used as populational markers in future surveys. The haplotypic diversity in major ethnic groups revealed high levels in Greater Asians, followed by Africans and Caucasians, and a very low diversity was seen in Amerindians. The discrimination probability of such haplotypes for a random sample of Brazilian Caucasians was 0.82, suggesting great potential usefulness in forensic studies. The parsimonious relationship between different alpha(h) types and the addition of the YAP polymorphism data allowed the construction of an informative picture of the origin and evolution of the antiquity of the alpha(h) polymorphism. The DYS19 allele diversity related to each alpha(h) type allowed a crude estimation of the antiquity of many alpha(h) types. These ancient alpha(h) types were present in different populations suggesting a common ancestor that could antedate the first out-of-Africa migrations.
Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content.
Authors: Baskaran N , Kandpal R P , Bhargava A K , Glynn M W , Bale A , Weissman S M
Published: June 2007

A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaq1 and Pfu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.
Direct cDNA selection with DNA microdissected from mouse chromosome 16: isolation of novel clones and construction of a partial transcription map of the C3-C4 region.
Authors: Wei J , Hodes M E , Wang Y , Feng Y , Ghetti B , Dlouhy S R
Published: June 2007

A group of cDNA segments was selected by direct hybridization of mouse cerebellar cDNAs against genomic DNA pools generated by microdissection of the mouse chromosome 16 (MMU16) C3-C4 region. After elimination of repetitive sequences and adjustment for redundancy among clones, 34 novel cDNA fragments were isolated. The MMU16 origin of clones was confirmed by genetic linkage mapping. Reverse transcription PCR indicated that approximately 68% of the cDNAs represent transcripts that are expressed in adult mouse cerebellum. Northern blotting showed that some of these are predominantly or solely expressed in brain. This work demonstrates that DNA microdissected from banded MMU16 can be used for direct cDNA selection, thus enabling construction of a new, region-specific partial transcription map. This selected cDNA library should be a useful reagent for further molecular neurobiological studies.
Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1Mb.
Authors: Wang Z F , Krasikov T , Frey M R , Wang J , Matera A G , Marzluff W F
Published: June 2007

The histone gene cluster on mouse chromosome 13 has been isolated and characterized. Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined. It contains 45 histone genes, organized in three patches containing tightly clustered genes. An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes. The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster. All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined. Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein. They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.
Characterization of the 55-kb mouse histone gene cluster on chromosome 3.
Authors: Wang Z F , Tisovec R , Debry R W , Frey M R , Matera A G , Marzluff W F
Published: June 2007

The histone gene cluster on mouse chromosome 3 has been isolated as a series of overlapping P1 clones, covering 110-120 kb, by probing with the histone H3-614 gene that had been mapped previously to mouse chromosome 3. There are genes for 10 core histone proteins present in a 55-kb cluster within this contig. There are three histone H3 genes, two of which are identical; four histone H2a genes, two of which are identical, one histone H4 gene; and two histone H2b genes. These histone H3 and H2a genes encode approximately 40% of the total H3 and H2a mRNA, whereas the histone H4 and histone H2b genes encode < 10% of the total H4 and H2b mRNA. There are no histone H1 genes present in this cluster. All of the histone H2a genes encode histone H2a.2 proteins (or variants of H2a.2), and account for all the H2a.2 genes in the mouse genome. All three histone H3 genes encode the histone H3.2 protein. A 21-kb region containing the adjacent H3-614 and H2a-614 genes has been duplicated and is present in an inverted repeat separated by 4.5 kb. The other two H2a genes are adjacent, with the 3' ends of their mRNAs separated by only 49 nucleotides in the DNA and the U7 snRNP binding sites separated by only 20 nucleotides. One of the histone H2b genes has lost the stem-loop sequence characteristic of the replication-dependent histone mRNAs and encodes only polyadenylated mRNAs.
Evidence for linkage of regions on chromosomes 6 and 11 to plasma glucose concentrations in Mexican Americans.
Authors: Stern M P , Duggirala R , Mitchell B D , Reinhart L J , Shivakumar S , Shipman P A , Uresandi O C , Benavides E , Blangero J , O'Connell P
Published: June 2007

The genetic factors involved in type II diabetes are still unknown. To address this problem, we are creating a 10 to 15 cM genetic map on 444 individuals from 32 Mexican American families ascertained on a type II diabetic proband. Using highly polymorphic microsatellite markers and a multipoint variance components method, we found evidence for linkage of plasma glucose concentration 2 hr after oral glucose administration to two regions on chromosome 11: beta-hemoglobin (HBB) and markers D11S899/D11S1324 near the sulfonylurea receptor (SUR) gene. Iod scores at these two loci were 2.77 and 3.37, respectively. The SUR gene region accounted for 44.7% of the phenotypic variance. Evidence for linkage to fasting glucose concentration was also observed for two loci on chromosome 6, one of which is identical to a proposed susceptibility locus for type I diabetes (D6S290). When diabetics were excluded from the analyses, all Iod scores became zero, suggesting that the observed linkages were with the trait diabetes rather than with normal variation in glucose levels. Results were similar whether all diabetics were included in the analyses or only those who were not under treatment with oral antidiabetic agents or insulin.
Toward the development of a gene index to the human genome: an assessment of the nature of high-throughput EST sequence data.
Authors: Aaronson J S , Eckman B , Blevins R A , Borkowski J A , Myerson J , Imran S , Elliston K O
Published: June 2007

A rigorous analysis of the Merck-sponsored EST data with respect to known gene sequences increases the utility of the data set and helps refine methods for building a gene index. A highly curated human transcript data base was used as a reference data set of known genes. A detailed analysis of EST sequences derived from known genes was performed to assess the accuracy of EST sequence annotation. The EST data was screened to remove low-quality and low-complexity sequences. A set of high-quality ESTs similar to the transcript data base was identified using BLAST; this subset of ESTs was compared with the set of known genes using the Smith-Waterman algorithm. Error rates of several types were assessed based on a flexible match criterion defining sequence identity. The rate of lane-tracking errors is very low, approximately 0.5%. Insert size data is accurate within approximately 20%. Reversed clone and internal priming error rates are approximately 5% and 2.5%, respectively, contributing to the incorrect identification of reads as 3' ends of genes. Follow-up investigation reveals that a significant number of clones, miscategorized as reversed, represent overlapping genes on the opposite strand of entries in the transcript data base. Relevance of these results to the creation of a high-quality index to the human genome capable of supporting diverse genomic investigations is discussed.
Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences.
Authors: Makałowski W , Zhang J , Boguski M S
Published: June 2007

A large set of mRNA and encoded protein sequences, from orthologous murine and human genes, was compiled to analyze statistical, biological, and evolutionary properties of coding and noncoding transcribed sequences. Protein sequence conservation varied between 36% and 100% identity, with an average value of 85%. The average degree of nucleotide sequence identity for the corresponding coding sequences was also approximately 85%, whereas 5' and 3' untranslated regions (UTRs) were less conserved, with aligned identities of 67% and 69%, respectively. For some mouse and human genes, nucleotide sequences are more highly conserved than the encoded protein sequences. A subset of 32 sequences, consisting of only mouse/human protein pairs for which the human sequence represents a positionally cloned disease gene, had properties very similar to the larger data set, suggesting that our data are representative of the genome as a whole. With respect to sequence conservation, two interesting outliers are the breast cancer (BRCAI) gene product and the testis-determining factor (SRY), both of which display among the lowest degrees of sequence identity. The occurrence of both introns and repetitive elements (e.g., Alu, Bl) in 5' and 3' UTRs was also studied. These results provide one benchmark for the "comparative genomics" of mice and humans, with practical implications for the cross-referencing of transcript maps. Also, they should prove useful in estimating the additional sampling diversity provided by mouse EST sequencing projects designed to complement the existing human cDNA collection.
Intron 17 of the human retinoblastoma susceptibility gene encodes an actively transcribed G protein-coupled receptor gene.
Authors: Herzog H , Darby K , Hort Y J , Shine J
Published: June 2007

The human retinoblastoma susceptibility gene, a member of the tumor suppressor gene family, is located on chromosome 13q14.12-13q14.2 and consists of 27 exons that are distributed over 180 kb. This study shows that intron 17, the largest in size, consisting of nearly 72,000 bp, contains an open reading frame encoding a novel G protein-coupled receptor in the reverse orientation relative to the transcription of the retino-blastoma susceptibility gene. Correction of a frameshift mutation revealed that this novel G protein-coupled receptor is the human homolog of a chicken T-cell-specific receptor cDNA. This is an additional description of an actively transcribed protein-encoding gene positioned within an intron of another gene, suggesting that introns can have important structural functions.
Mutations in sheep microsatellites.
Authors: Crawford A M , Cuthbertson R P
Published: June 2007

During the construction of a primary linkage map of the sheep genome, a total of 46,225 parent-offspring transfer of microsatellite alleles were examined through the genotyping of the AgResearch International Mapping Flock. Any observations of mutations were thoroughly rechecked, including examination of new DNA samples, to yield five bona fide mutations from the study. This gave us an observed spontaneous mutation rate for microsatellites in sheep of 1.1 +/- 0.5 x 10(-4) mutations/gamete per locus. Untransformed lymphocytes were used as the source of DNA. Two of the mutations could have happened at either meiosis or during mitotic growth of the male germ cells. The other three mutations probably occurred early in the mitotic generation of the female germ cells. All of the mutations consisted of a 2-bp insertion and occurred in individuals heterozygous at the mutant locus.
Cloning of the murine homolog of the ocular albinism type 1 (OA1) gene: sequence, genomic structure, and expression analysis in pigment cells.
Authors: Bassi M T , Incerti B , Easty D J , Sviderskaya E V , Ballabio A
Published: June 2007

We report the isolation of the mouse homolog of OA1, the gene responsible for ocular albinism type 1. The mouse Oa1 gene encodes a putative protein of 405 amino acids displaying a high level of homology (78% identity, 87% similarity) to the human gene. All disease-associated missense mutations reported in patients with ocular albinism involve conserved amino acid residues in the mouse protein. Moreover, the murine homolog shows six putative transmembrane domains, as observed for the human gene, indicating that the overall structure of the two proteins is conserved. The genomic organization is also conserved between the two species across the entire coding region with splice sites located in the same positions. Like its human counterpart, the expression pattern of Oa1, apart from the eye, is restricted to the epidermal melanocyte lineage. A transcript of approximately 1.8 kb was readily detected by this probe in 5 out of 5 murine melanocyte lines, 4 out of 4 murine melanoblast lines, 1 out of 2 murine melanoma lines, and 1 out of 2 human melanoma lines tested, but it was not detected in 2 out of 2 lines of a developmentally earlier normal cell type, melanoblast precursor cells, suggesting that the gene is transcriptionally activated in epidermal melanocytes at the same stage as most other tested melanosomal proteins. Together, these data suggest that the function of the OA1 gene is conserved between human and mouse and point to the mouse as a model to facilitate the understanding of ocular albinism pathogenesis.
Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis.
Authors: Mansfield E S , Vainer M , Enad S , Barker D L , Harris D , Rappaport E , Fortina P
Published: June 2007

The Human Genome Initiative has increased significantly the rate at which disease-causing genes are being mapped and sequenced. New cost-effective methods to locate the genes and to characterize disease-causing mutations require robust, reproducible, and accurate protocols for measuring DNA fragment lengths. Capillary array electrophoresis (CAE) offers rapid, high-resolution separations, high throughput, and sensitive detection. To assess the utility of CAE for the accumulation of genetic information, we tested both sizing accuracy and reproducibility using 48-capillary prototype systems. Two multiplex PCR allelic ladder standards and several CA-repeat markers were analyzed in > 100 runs. Reproducibility in typing > 8000 genotypes reveals a standard deviation of less than 0.2 bp on these systems under optimized conditions. However, sequence-dependent migration anomalies were observed at most simple sequence loci even when analyzed under denaturing conditions, resulting in a systematic bias in estimated fragment sizes. We show here that, by normalizing results to known typing controls, one can obtain locus-averaged accuracies of < 0.06 bp and normalized results within 1 bp of actual. We detect as little as a 1:30,000 dilution of a DNA quantitation standard stained with highly sensitive intercalating dyes, indicating an 80-zeptomole sensitivity limit. However, to obtain reproducible electrokinetic injection, approximately 200 attomoles of fluorescein-labeled DNA is required. These sensitivity limits, sizing precision, and accuracy, together with the 1-hr run times for 48-96 samples, indicate that CAE is a viable method for high-throughput genetic analysis of simple sequence repeat polymorphisms.
Mapping expressed sequence tag sites on yeast artificial chromosome clones of Arabidopsis thaliana DNA.
Authors: Agyare F D , Lashkari D A , Lagos A , Namath A F , Lagos G , Davis R W , Lemieux B
Published: June 2007

We describe a method for efficient parallel mapping of expressed sequence tag (EST) sites onto yeast artificial chromosome (YAC) clones. The strategy involves an initial YAC clone pooling scheme that minimizes the number of required PCR amplifications. This is followed by parallel analysis of PCR amplicons of EST sequences. Using this method, we have screened 600 EST sites in combinatorial pools of 3449 YAC clones that contain Arabidopsis thaliana DNA inserts. The presence of these genes on YACs was detected by amplifying EST sequences with PCR and analyzing the reaction products by agarose gel electrophoresis. Of the 600 ESTs, 271 were found to map to individual YACs. Software tools are presented that allow for the automated analysis of this electrophoresis data. Suggestions for the scale-up of this method to map large genomes are discussed.
4.5-Mb YAC STS contig at 50-kb resolution, spanning Xq25 deletions in two patients with lymphoproliferative syndrome.
Authors: Porta G , MacMillan S , Nagaraja R , Mumm S , Zucchi I , Pilia G , Maio S , Featherstone T , Schlessinger D
Published: June 2007

Sequence-tagged site (STS) content mapping in yeast artificial chromosomes (YACs) was used to cover the region deleted in two patients affected with X-linked lymphoproliferative disorder. The order of markers includes, centromere to telomere, DXS8009-DXS1206-DXS8078-DXS8044-DXS982- DXS6811-DXS8093-AFM240xblO- DXS75-DXS737-DXS100-DXS6-DXS1046-DXS803 8. The order of six major markers is confirmed by fluorescent in situ hybridization, and all the markers assigned by linkage mapping fall within a 1.6-cM interval. The contig comprises 90 clones containing 89 STSs, yielding a resolution of 50 kb; DNA in a gap just telomeric to DXS8044 has not been found in > 20 equivalents of YACs or bacterial clones. The two deletions were found to have centromeric breakpoints that lie close to DXS1206 and may be identical; the telomeric breakpoints are -150 kb apart, one falling between DXS737 and DXS100, the other between DXS100 and DXS1046. Several STSs near the breakpoints show weak amplification from more than one site; one gives products from three groups of YACs, and lie, respectively, within 50 kb of the centromeric and the two telomeric deletion borders. Such partially duplicated segments of DNA are candidates for involvement in the formation of the deletions.
A collection of 1814 human chromosome 7-specific STSs.
Authors: Bouffard G G , Iyer L M , Idol J R , Braden V V , Cunningham A F , Weintraub L A , Mohr-Tidwell R M , Peluso D C , Fulton R S , Leckie M P , Green E D
Published: June 2007

An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes. En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs. The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries. This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.
Incognito rRNA and rDNA in databases and libraries.
Authors: Gonzalez I L , Sylvester J E
Published: June 2007

Both ribosomal DNA (rDNA) and ribosomal RNA (rRNA) are over-represented in the starting material for genomic and cDNA libraries; thus, their sequences have the potential of repeatedly entering the various databases. When DNA (both transcribed and intergenic spacer regions) is used as query sequence, a great number of matches are found in the databases, particularly in the EST database, and to a lesser extent among genomic sequences and STSs, which are not identified as rDNA. We discuss the following explanations for the widespread occurrence of rDNA in cDNA and genomic DNA libraries: pseudogenes of rRNA in other genomic locations, mRNA-derived pseudogenes that reside in rDNA, cDNAs derived from rRNA [either by self-priming or by internal oligo(dT) priming], cDNAs derived from actual transcripts of the rDNA intergenic spacer, and genomic DNA contamination of RNA preparations. Because so many database entries contain unidentified rDNA, we recommend that all sequence submissions be checked (by the submitters) for the presence of structural RNAs in addition to repetitive sequences.
A chromosome-specific microdissected library increases marker density on bovine chromosome 1.
Authors: Sonstegard T S , Abel Ponce de León F , Beattie C W , Kappes S M
Published: June 2007

Genetic resolution of bovine chromosome 1 (BTA1) linkage group was significantly increased by screening for microsatellite clones a microdissected library constructed from a bovine cell line carrying a t(1;29) translocation. Eighty-five percent of the microsatellites (ms) (46/54) identified were informative in the USDA/MARC mapping population, and 96% of these ms (44/46) linked to BTA1 (LOD > 3.0). When merged with 40 existing BTA1 markers the genetic map spanned 153.8 cM (sex-averaged interval, 1.9 cM). The fourfold improvement in marker density of BTA1 provides a genetic map that enhances mapping of quantitative trait loci and implementation of marker assisted selection.
High-throughput microsatellite analysis using fluorescent dUTPs for high-resolution genetic mapping of the mouse genome.
Authors: Rhodes M , Dearlove A , Straw R , Fernando S , Evans A , Greener M , Lacey T , Kelly M , Gibson K , Brown S D , Mundy C
Published: June 2007

The use of fluorescent end-labeled primers has proved successful for rapid, semiautomated genotyping of microsatellite loci. However, custom synthesis is expensive and costs can be prohibitive when a wide range of markers is to be analyzed for only a few genotypings. This particularly applies to high-resolution genetic mapping in the mouse either in the construction of global maps or in the production of local high-resolution genetic maps for positional cloning. We demonstrate here the use of fluorescent dUTPs for cost-effective, high-throughput microsatellite genotyping in the mouse. This alternative to the use of fluorescent end-labeled primers for semiautomated genotyping is potentially applicable to the construction of linkage maps in other species.
Isolation of cDNAs from the Cri-du-chat critical region by direct screening of a chromosome 5-specific cDNA library.
Authors: Simmons A D , Overhauser J , Lovett M
Published: June 2007

Chromosome-specific cDNA libraries are new tools for the isolation of genes from specific genomic regions. We have used two YACs than span the approximately 2-Mb cri-du-chat critical region (CDCCR) of chromosome 5p to directly screen a chromosome 5-specific (CH5SP) fetal brain cDNA library. To compare this library with other sources for new gene discovery, the YACs were hybridized to a normalized infant brain (NIB) cDNA library that has been used extensively for expressed sequence tag (EST) generation. These screens yielded 12 cDNAs from the CH5SP fetal brain library and four cDNAs from the NIB library that mapped to discrete intervals within the CDCCR. Four cDNAs mapped within the minimal CDCCR deletion interval, with the remaining cDNAs being located beyond the boundaries. Only one cDNA shared sequence overlap between the CH5SP and NIB sets of clones. None of the remaining 11 CH5SP cDNAs were homologous to EST sequences, suggesting, in common with previous data on these libraries, that chromosome-specific cDNA libraries are a rich source of new expressed sequences. The single cDNA that did overlap with the NIB library contained two copies of a sequence motif shared with thrombospondin, properdin, and several complement proteins. This motif is usually present in adhesive proteins, and appears to mediate cell-cell or cell-substrate interactions. This new thrombospondin-like gene, and the other three cDNAs that map within the CDCCR, represent candidate genes for the cri-du-chat contiguous gene deletion syndrome.
Cloning and characterization of two vertebrate homologs of the Drosophila eyes absent gene.
Authors: Zimmerman J E , Bui Q T , Steingrímsson E , Nagle D L , Fu W , Genin A , Spinner N B , Copeland N G , Jenkins N A , Bucan M , Bonini N M
Published: June 2007

The Drosophila eyes absent (eya) gene plays an essential role in the events that lead to proper development of the fly eye and embryo. Here we report the analysis of two human and two mouse homologs of the fly eya gene. Sequence comparison reveals a large domain of approximately 270 amino acids in the carboxyl terminus of the predicted mammalian proteins that shows 53% identity between the fly sequence and all of the vertebrate homologs. This Eya-homology domain is of novel sequence, with no previously identified motifs. RNA hybridization studies indicate that the mouse genes are expressed during embryogenesis and in select tissues of the adult. Both mouse Eya genes are expressed in the eye, suggesting that these genes may function in eye development in vertebrates as eya does in the fly. The mouse Eya2 gene maps to chromosome 2 in the region syntenic with human chromosome 20q13, and the mouse Eya2 gene maps to chromosome 4 in the region syntenic with human chromosome 1p36. Our findings support the notion that several families of genes (Pax-6/eyeless, Six-3/sine oculis, and Eya) play related and critical roles in the eye for both files and vertebrates.
Retrofitting vectors for Escherichia coli-based artificial chromosomes (PACs and BACs) with markers for transfection studies.
Authors: Mejía J E , Monaco A P
Published: June 2007

P1-based artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have significantly expanded the size of fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules. Functional characterization of a gene within a given PAC or BAC clone often requires transferring the DNA into eukaryotic cells for transient or long-term expression. To facilitate transfection studies, we have developed protocols using the Notl restriction sites of any PAC or BAC clone to introduce a transfection reporter gene, lacZ, together with a selectable marker, neo. This enables transfected cells to be detected by X-Gal staining to verify DNA uptake, and clones of stably transformed cells may be selected for in the presence of the antibiotic G418. The same retrofitting protocols may be applied with other markers of interest to extend the functionality of PAC and BAC libraries, and specialized aspects of such manipulation of E. coli-based artificial chromosomes are outlined.
Confirmation of quantitative trait loci for ethanol sensitivity in long-sleep and short-sleep mice.
Authors: Markel P D , Bennett B , Beeson M , Gordon L , Johnson T E
Published: June 2007

Initial insensitivity to alcohol is a strong predictor of human alcoholism, a widespread and heritable health problem. The Long Sleep and Short Sleep lines of mice were developed by genetic selection for high or low alcohol sensitivity. We have identified seven quantitative trait loci (QTLs) specifying differences in alcohol sensitivity using intercross progeny from these selected strains. These QTLs (Lorel-Lore7) together account for approximately 60% of the total genetic variance for this trait. This represents the first report of linkages for genes influencing alcohol action in any mammalian system using stringent, genome-wide mapping criteria.
Genome DNA sequencing around the EF-1 alpha multigene locus of Arabidopsis thaliana indicates a high gene density and a shuffling of noncoding regions.
Authors: Tremousaygue D , Bardet C , Dabos P , Regad F , Pelese F , Nazer R , Gander E , Lescure B
Published: June 2007

In Arabidopsis thaliana, EF-1 alpha proteins are encoded by a multigene family of four members. Three of them are clustered at the same locus, which was positioned 24 cM from the top of chromosome 1. A region of DNA spanning 63 kb around these locus was sequenced and analyzed. One main characteristic of the locus is the mosaic organization of both genes and intergenic regions. Fourteen genes were identified, among which only four were already described, and other unidentified are most likely present. Functionally diverse genes are found at close intervals. Exon and intron distribution is highly variable at this locus, one gene being split into at least 20 introns. Several duplications were found within the sequenced segment both in coding and noncoding regions, including two gene families. Moreover, a sequence corresponding to the 5' noncoding region of the EF-1 alpha genes and harboring a 5' intervening sequence is duplicated and found upstream of several genes, suggesting that noncoding regions can be shuffled during evolution.
Identification of the complete coding sequence and genomic organization of the Treacher Collins syndrome gene.
Authors: Dixon J , Edwards S J , Anderson I , Brass A , Scambler P J , Dixon M J
Published: June 2007

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Recently, the demonstration of a series of 10 mutations within a partial-length cDNA clone have indicated that the TCS gene (TCOF1) has been positionally cloned. Although it has been shown that the gene is expressed in a wide variety of fetal and adult tissues, database sequence comparisons have failed to provide significant information on the function of the gene. In the current investigation, a combination of cDNA library screening and rapid amplification of cDNA ends has permitted the isolation of the complete coding sequence of TCOF1, which is encoded by 26 exons and predicts a low complexity, serine/alanine-rich protein of approximately 144 kD. The use of a variety of bioinformatics tools has resulted in the identification of repeated units within the gene, each of which maps onto an individual exon. The predicted protein Treacle contains numerous potential phosphorylaiton sites, a number of which map to similar positions within the repeated units, and shows weak but significant homology to the nucleolar phosphoproteins. Although the precise function of Treacle remains unknown, these observations suggest that phosphorylation may be important for its role in early embryonic development and that it may play a role in nucleolar-cytoplasmic shuttling. The information presented in this study will allow continued mutation analysis in families with a history of TCS and should facilitate continued experimentation to shed further light on the function of the gene/protein during development of the craniofacial complex.
A second-generation linkage map of the bovine genome.
Authors: Kappes S M , Keele J W , Stone R T , McGraw R A , Sonstegard T S , Smith T P , Lopez-Corrales N L , Beattie C W
Published: June 2007

We report a bovine linkage map constructed with 1236 polymorphic DNA markers and 14 erythrocyte antigens and serum proteins. The 2990-cM map consists of a sex-specific, X chromosome linkage group and 29 sex-averaged, autosomal linkage groups with an average interval size of 2.5 cM. The map contains 627 new markers and 623 previously linked markers, providing a basis for integrating the four published bovine maps. Orientation and chromosomal assignment of all the linkage groups, except BTA20 and BTA22, was provided by 88 markers that were assigned previously to chromosomes. This map provides sufficient marker density for genomic scans of populations segregating quantitative trait loci (QTL) and subsequent implementation of marker-assisted selection (MAS) mating schemes.
Overexpression of a calcium-binding protein, S100 beta, in astrocytes alters synaptic plasticity and impairs spatial learning in transgenic mice.
Authors: Gerlai R , Wojtowicz J M , Marks A , Roder J
Published: June 2007

Recent evidence suggests that slowly propagating Ca2+ waves from astrocytes can modulate the function of neurons. Altering astrocytic calcium processes in vivo may therefore affect neuronal and behavioral phenotypes. Previously, we generated transgenic mice that overexpress an astrocytic calcium-binding protein, S100 beta. Immunocytochemistry and in situ hybridization showed elevated expression in the astrocytes of the hippocampus and other brain regions. Neurons in the hippocampus were negative for S100 beta. In this paper we analyze the hippocampal electrophysiology and learning properties of mice from two transgenic lines. Significant differences were found between the hippocampal slices of normal and transgenic mice in their response to high frequency (100 Hz) stimulation. The overall distribution of post-tetanic excitatory postsynaptic potentials (EPSP) of the slices from the transgenic mice was shifted significantly toward smaller values to a degree that 25% of slices exhibited depression. The altered hippocampal neurophysiology was accompanied by an impairment in a hippocampal-dependent learning task. Transgenic mice showed significant impairment in a spatial version of the Morris water maze, however, they performed normally in non-spatial tasks. Probe trials showed that transgenic mice, though significantly impaired, also acquired spatial information. The results suggested that the impairment was not due to motor dysfunction, impaired vision or motivation of the transgenic mice, findings compatible with a possible hippocampal mechanism. We conclude that overexpression of S100 beta in astrocytes impairs, but does not abolish, the ability to solve a spatial task, and it leads to a significantly decreased post-tetanic potentiation in the hippocampal slice. We hypothesize that the changes are due to calcium mediated processes. Our results support the notion that astrocytes are involved in higher brain functions.
Environmental enrichment and postnatal handling prevent spatial learning deficits in aged hypoemotional (Roman high-avoidance) and hyperemotional (Roman low-avoidance) rats.
Authors: Escorihuela R M , Tobeña A , Fernández-Teruel A
Published: June 2007

The present study investigated the enduring effects of postnatal handling (administered during the first 21 days of life), and environmental enrichment (for a period of 6 months starting 3 weeks after weaning) on spatial learning in 24-month-old hypoemotional (Roman high-avoidance, RHA/Verh) and hyperemotional (Roman low-avoidance, RLA/Verh) rats. Two groups of 5-month-old rats from both lines were also included in the experiment as young controls. The Roman lines performed differently in the Morris water maze: Path lengths of RLA/Verh rats were shorter and they swam at lower speed than RHA/Verh rats, showing quicker and more efficient learning overall. Postnatal handling improved learning mainly in RHA/Verh rats, whereas environmental enrichment was able to prevent the deficits shown by aged controls of both lines. Young, enriched, and handled plus enriched animals exhibited better performance than impaired aged controls, to the point that aged enriched and handled plus enriched animals did not differ from young controls. Thus, besides indicating that RLA/Verh rats are better learners than RHA/Verh rats in the Morris water maze, this study demonstrates that environmental enrichment prevents the cognitive loss associated with aging, over the long term. Finally, the positive effects obtained with postnatal handling were dependent on the rat line.
Analysis of conditioned courtship in dusky-Andante rhythm mutants of Drosophila.
Authors: van Swinderen B , Hall J C
Published: June 2007

Genetic connections between learning and rhythmicity were suggested to have been established in a previous study, in part because the duskyAndante (dyAnd) mutation in Drosophila disrupted both behaviors. dyAnd, isolated as a slow-clock variant, was reported to cause an approximately fourfold decrement in courtship-suppression conditioning. These effects have been reexamined; the experiments were buttressed by testing the effects of several recently isolated mutations at the dusky locus, along with the original And Allele that had been induced there. The reexamination was also prompted by anatomical concerns, certain of which have recently focused on dy-induced decrements in cell size, but only in terms of wing morphology. Another anatomical issue involves the discovery of a neuronal pathway that seems to connect circadian pacemaker cells to a structure in the Drosophila brain that is involved in learning. In observer-blind experiments, however, it was found that neither pacemaker-slowing (Andante-like) dy mutations nor others that cause no rhythm defects produced subnormal conditioned courtship. Moreover, in the adult brain of a slow-clock dyAnd mutant, no axonal pathway defects were readily discernible and putative pacemaker neurons appeared to be normal in size.
Integration of olfactory information in a spatial representation enabling accurate arm choice in the radial arm maze.
Authors: Lavenex P , Schenk F
Published: June 2007

The aim of the present study was to determine whether and how rats can use local olfactory cues for spatial orientation. Rats were trained in an eight-arm radial maze under different conditions as defined by the presence or absence of supplementary olfactory cues marking each arm, the availability of distant visuospatial information, and the illumination of the maze (light or darkness). The different visual conditions were designed to dissociate among the effects of light per se and those of visuospatial cues, on the use of olfactory cues for accurate arm choice. Different procedures with modifications of the arrangement of olfactory cues were used to determine if rats formed a representation of the spatial configuration of the olfactory cues and if they could rely on such a representation for accurate arm choice in the radial maze. The present study demonstrated that the use of olfactory cues to direct arm choice in the radial arm maze was critically dependent on the illumination conditions and implied two different modes of processing of olfactory information according to the presence or the absence of light. Olfactory cues were used in an explicit manner and enabled accurate arm choice only in the absence of light. Rats, however, had an implicit memory of the location of the olfactory cues and formed a representation of the spatial position of these cues, whatever the lighting conditions. They did not memorize the spatial configuration of the olfactory cues per se but needed these cues to be linked to the external spatial frame of reference.
c-Fos induction in the rat nucleus of the solitary tract correlates with the retention and forgetting of a conditioned taste aversion.
Authors: Houpt T A , Philopena J M , Joh T H , Smith G P
Published: June 2007

Recently, we have described a potential neuronal correlate of the behavioral expression of a conditioned taste aversion (CTA) against sucrose at the level of c-Fos expression. Intraoral infusions of sucrose induce c-Fos-like immunoreactivity (c-FLI) in the intermediate nucleus of the solitary tract (iNTS) after a CTA has been acquired for sucrose. Sucrose infusions do not induce c-FLI in the iNTS of unconditioned rats or in conditioned rats after extinction of the CTA. Here, we describe persistence of altered responsiveness of the iNTS in rats with CTAs against sucrose by intraorally infusing sucrose 2 days, 3 months, or 6 months after acquisition of the CTA. Sucrose infusions induced c-FLI in the iNTS 6 months after conditioning. The behavioral expression of the CTA was attenuated at 6 months but not at 3 months; the number of c-FLI positive cells in the iNTS was proportional to the magnitude of the expression of the CTA. This evidence strengthens our hypothesis that c-FLI in the iNTS is a neuronal correlate of the expression of a CTA.