bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligation-mediated PCR of restriction fragments from large DNA molecules.
Authors: Smith D R
Published: June 2007

A general method is described for PCR amplification of single restriction fragments from large DNA molecules. The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases. Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions. The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease. The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target. Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently. Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments. In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags.
Automated DNA profiling by fluorescent labeling of PCR products.
Authors: Sullivan K M , Pope S , Gill P , Robertson J M
Published: June 2007

DNA profiling has been automated by the fluorescent tagging of amplified variable number tandem repeat (VNTR) loci. This was achieved by the use of fluorescently labeled primers in the amplification of 10 ng of genomic DNA, coupled with laser detection of the products during electrophoresis. The PCR products are sized by co-electrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation caused by lane-to-lane differences in migration rate. This increases the precision of VNTR characterization and enables alleles that differ by a single 15-bp repeat to be resolved. The system is capable of high throughput: Twenty-four samples are electrophoresed and analyzed within 6 hr. Also, because four different dyes are available, three different loci can be simultaneously characterized with the fourth dye used for the internal standard. Approximately 100 unrelated British caucasians were analyzed at the loci D1S80, D17S5, and ApoB. The probabilities of two unrelated individuals matching by chance (pM) at these three loci were determined to be 0.065, 0.040, and 0.069, respectively, with a combined pM of 1.8 x 10(-4).
The use of hybrid-selected template increases the specificity of the polymerase chain reaction.
Authors: Lucas W T , Youngner J S
Published: June 2007

An efficient method for generating full-length DNA sequences from apparently unsuccessful polymerase chain reactions (PCR) has been developed. In cases where nonspecific background interferes with detection of the PCR product, a second amplification is performed using a nested set of primers. The internal fragment of DNA amplified in this reaction is then blotted to a membrane and used to hybrid-select the desired DNA from the initial amplification. This DNA is eluted and used as the template for a third round of PCR. The re-use of the original primers from the initial reaction enables the final PCR to generate full-length DNA. This technique was used to clone a full-length gene segment 8 from a mutant influenza A/WSN/33 (H1N1) virus after initial PCR attempts had failed.
A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase.
Authors: Rashtchian A , Thornton C G , Heidecker G
Published: June 2007

A novel method for site-directed mutagenesis of DNA sequences based on the use of the PCR is described. The method uses two oligonucleotide primers that contain the desired sequence change and overlap at their 5' ends. In addition, the thymine residues in the overlap region have been substituted with deoxyuracil. Amplification of the template plasmid by PCR results in incorporation of the primers and the desired mutation into the PCR product. Excision of the deoxyuracil residues in the PCR products by uracil DNA glycosylase (UDG) destablizes base-pairing at the ends of DNA molecules and thus generates 3' protruding ends in the opposite strand. Due to overlapping nature of the primers, the resulting 3' protruding ends are complementary and can anneal rapidly after treatment with UDG. When the entire plasmid is amplified, a linear mutant PCR product is generated that circularizes after treatment with UDG. Circularized molecules can then be transformed into competent cells without ligation, generating transformants with the mutant genotype. Alternatively, the gene of interest is amplified in two segments using overlapping mutant primers and cloned in the desired orientation into pAMP1 by UDG cloning. Application of this method to site-specific mutagenesis of the lacZ alpha gene and the human c-raf oncogene was demonstrated. The accuracy of the mutations was confirmed by nucleotide sequence analysis as well as phenotypic assays. The method is rapid, highly efficient (> 99%), and applicable to genes cloned in any vector as well as to genomic DNA or RNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Development of a sensitive reverse transcriptase PCR assay, RT-RPCR, utilizing rapid cycle times.
Authors: Tan S S , Weis J H
Published: June 2007

We describe a procedure to analyze rare gene transcripts quantitatively using a reverse transcriptase-polymerase chain (RT-PCR) reaction. RNA purified from cells and tissues is reverse-transcribed using random hexamer primers and is amplified using very short cycle times. The products are trace-labeled with [32P[dCTP, which allows for the quantitation of the products by gel electrophoresis and excision of bands. The quantity of the PCR product is directly proportional to the quantity of cDNA added and is reproducible from a single cDNA preparation or from samples derived from separate preparations. Because cDNA synthesis is primed with random oligonucleotides, the same cDNA sample preparation can be examined for many different gene products.
Partial endonuclease digestion mapping of restriction sites using PCR-amplified DNA.
Authors: Morales J C , Patton J C , Bickham J W
Published: June 2007

Although direct DNA sequencing is now readily available, restriction enzyme analyses are still widely used in population genetics and molecular systematics studies. These analyses provide cheaper and faster ways to assay patterns of nucleotide differentiation across a large number of individuals. In this paper, we introduce a new approach to restriction enzyme analyses in which high-resolution restriction site maps are obtained from partial digestions of PCR products. This procedure increases the level of resolution at least an order of magnitude over the double-digestion method for restriction enzyme mapping, can target specific DNA regions with the use of specific primers, and, because it uses chemiluminescent detection of DNA, can be easily implemented in laboratories that lack the necessary setups to handle radioactive substances.
Importance of different variables for enhancing in situ detection of PCR-amplified DNA.
Authors: Nuovo G J , Gallery F , Hom R , MacConnell P , Bloch W
Published: June 2007

This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells. A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion. A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution. This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin. The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein. The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions.
Mechanically stretched chromosomes as targets for high-resolution FISH mapping.
Authors: Laan M , Kallioniemi O P , Hellsten E , Alitalo K , Peltonen L , Palotie A
Published: June 2007

When used with metaphase chromosomes, fluorescence in situ hybridization (FISH) makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by > or = 2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the 1p32-p33 region, spanning distances from 20 to approximately 1500 kb. Probes separated by > or = 170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from < 10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COL9A2, RLF, and L-MYC genes at 1p32. The use of mechanically stretched chromosomes provides < or = 10-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping (1-300 kb) techniques, such as the DNA fiber FISH.
Genes and languages in Europe: an analysis of mitochondrial lineages.
Authors: Sajantila A , Lahermo P , Anttinen T , Lukka M , Sistonen P , Savontaus M L , Aula P , Beckman L , Tranebjaerg L , Gedde-Dahl T , Issel-Tarver L , DiRienzo A , Pääbo S
Published: June 2007

When mitochondrial DNA sequence variation is analyzed from a sample of 637 individuals in 14 European populations, most populations show little differentiation with respect to each other. However, the Saami distinguish themselves by a comparatively large amount of sequence difference when compared with the other populations, by a different distribution of sequence diversity within the population, and by the occurrence of particular sequence motifs. Thus, the Saami seem to have a long history distinct from other European populations. Linguistic affiliations are not reflected in the patterns of relationships of mitochondrial lineages in European populations, whereas prior studies of nuclear gene frequencies have shown a correlation between genetic and linguistic evolution. It is argued that this apparent contradiction is attributable to the fact that genetic lineages and gene frequencies reflect different time perspectives on population history, the latter being more in concordance with linguistic evolution.
The human obese (OB) gene: RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome 7.
Authors: Green E D , Maffei M , Braden V V , Proenca R , DeSilva U , Zhang Y , Chua S C , Leibel R L , Weissenbach J , Friedman J M
Published: June 2007

The recently identified mouse obese (ob) gene apparently encodes a secreted protein that may function in the signaling pathway of adipose tissue. Mutations in the mouse ob gene are associated with the early development of gross obesity. A detailed knowledge concerning the RNA expression pattern and precise genomic location of the human homolog, the OB gene, would facilitate examination of the role of this gene in the inheritance of human obesity. Northern blot analysis revealed that OB RNA is present at a high level in adipose tissue but at much lower levels in placenta and heart. OB RNA is undetectable in a wide range of other tissues. Comparative mapping of mouse and human DNA indicated that the ob gene is located within a region of mouse chromosome 6 that is homologous to a portion of human chromosome 7q. We mapped the human OB gene on a yeast artificial chromosome (YAC) contig from chromosome 7q31.3 that contains 43 clones and 19 sequence-tagged sites (STSs). Among the 19 STSs are eight corresponding to microsatellite-type genetic markers, including seven (CA)n repeat-type Genethon markers. Because of their close physical proximity to the human OB gene, these eight genetic markers represent valuable tools for analyzing families with evidence of hereditary obesity and for investigating the possible association between OB mutations and human obesity.
Regional assignment of 68 new human gene transcripts on chromosome 11.
Authors: Rosier M F , Reguigne-Arnould I , Couillin P , Devignes M D , Auffray C
Published: June 2007

We have tested 80 expressed sequence-tagged site (eSTS) markers assigned to human chromosome 11 by the Genexpress program on a panel of somatic cell hybrids containing parts of this chromosome, characterized by cytogenetic data, reference markers, and with respect to the Généthon microsatellite genetic map. Sixty-eight new gene transcripts have been assigned to 25 subregions, one of which was newly defined by five of the eSTS markers. The markers are distributed on the short and long arms in agreement with their physical length. The genic map thus obtained has been integrated with the cytogenetic, genetic, and disease maps. Two eSTS markers have been further mapped with respect to a yeast artificial chromosome (YAC) contig close to the brain-derived neurotrophic factor (BDNF) gene and thus provide potential candidate genes for the mental retardation phenotype of WAGR (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) syndrome. Altogether, the 68 new gene transcripts localized here represent more than a threefold increase in the number of unknown regionalized genes that could reveal potential candidate genes for the numerous orphan pathologies associated with chromosome 11.
The organization of the human immunoglobulin lambda gene locus.
Authors: Kawasaki K , Minoshima S , Schooler K , Kudoh J , Asakawa S , de Jong P J , Shimizu N
Published: June 2007

To elucidate the complex structure of the human immunoglobulin lambda gene locus, a 1020-kb contig was constructed using 184 cosmid clones and one bacterial artificial chromosome (BAC) clone. A high-resolution physical map of this contig revealed that the entire lambda gene locus is 911 kb in length. It contains seven constant region (C lambda) gene segments and 69 unique EcoRI-HindIII segments that hybridize to variable region gene (V lambda) probes. The VpreB gene, BCRL4, and gamma-glutamyl transpeptidase gene (GGT)-like sequences are also located within the lambda gene locus. Hybridization analysis suggested that the lambda gene locus has undergone extensive amplification events in evolution.
BEAUTY: an enhanced BLAST-based search tool that integrates multiple biological information resources into sequence similarity search results.
Authors: Worley K C , Wiese B A , Smith R F
Published: June 2007

BEAUTY (BLAST enhanced alignment utility) is an enhanced version of the NCBI's BLAST data base search tool that facilitates identification of the functions of matched sequences. We have created new data bases of conserved regions and functional domains for protein sequences in NCBI's Entrez data base, and BEAUTY allows this information to be incorporated directly into BLAST search results. A Conserved Regions Data Base, containing the locations of conserved regions within Entrez protein sequences, was constructed by (1) clustering the entire data base into families, (2) aligning each family using our PIMA multiple sequence alignment program, and (3) scanning the multiple alignments to locate the conserved regions within each aligned sequence. A separate Annotated Domains Data Base was constructed by extracting the locations of all annotated domains and sites from sequences represented in the Entrez, PROSITE, BLOCKS, and PRINTS data bases. BEAUTY performs a BLAST search of those Entrez sequences with conserved regions and/or annotated domains. BEAUTY then uses the information from the Conserved Regions and Annotated Domains data bases to generate, for each matched sequence, a schematic display that allows one to directly compare the relative locations of (1) the conserved regions, (2) annotated domains and sites, and (3) the locally aligned regions matched in the BLAST search. In addition, BEAUTY search results include World-Wide Web hypertext links to a number of external data bases that provide a variety of additional types of information on the function of matched sequences. This convenient integration of protein families, conserved regions, annotated domains, alignment displays, and World-Wide Web resources greatly enhances the biological informativeness of sequence similarity searches. BEAUTY searches can be performed remotely on our system using the "BCM Search Launcher" World-Wide Web pages (URL is < http:/ /gc.bcm.tmc.edu:8088/ search-launcher/launcher.html > ).
A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.
Authors: Torigoe K , Sato S , Kusaba H , Kohno K , Kuwano M , Okumura K , Green E D , Tsui L C , Scherer S W , Schlessinger D
Published: June 2007

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs. A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones. The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes. Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others.
The Genexpress Index: a resource for gene discovery and the genic map of the human genome.
Authors: Houlgatte R , Mariage-Samson R , Duprat S , Tessier A , Bentolila S , Lamy B , Auffray C
Published: June 2007

Detailed analysis of a set of 18,698 sequences derived from both ends of 10,979 human skeletal muscle and brain cDNA clones defined 6676 functional families, characterized by their sequence signatures over 5750 distinct human gene transcripts. About half of these genes have been assigned to specific chromosomes utilizing 2733 eSTS markers, the polymerase chain reaction, and DNA from human-rodent somatic cell hybrids. Sequence and clone clustering and a functional classification together with comprehensive data base searches and annotations made it possible to develop extensive sequence and map cross-indexes, define electronic expression profiles, identify a new set of overlapping genes, and provide numerous new candidate genes for human pathologies.
A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase.
Authors: Byrappa S , Gavin D K , Gupta K C
Published: June 2007

Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.
Assessment of a mutation in the H5 domain of Girk2 as a candidate for the weaver mutation.
Authors: Mjaatvedt A E , Cabin D E , Cole S E , Long L J , Breitwieser G E , Reeves R H
Published: June 2007

A mutation in the GIRK2 inwardly rectifying K+ channel was mapped recently to the region of mouse chromosome 16 containing the wv gene and shown to occur in mutant but not in wild-type mice. We demonstrate tight linkage of the Girk2 mutation to the wv phenotype and refine the localization of the weaver (wv) gene on recombinational and physical maps. This linkage between Girk2 and wv has existed since at least 1988 in descendants of the original mutation maintained in C57BL/6 animals. Girk2 is shown to be transcribed in brain before the first recognized manifestation of the wv phenotype and in cultures of granule cells (GCs) isolated from cerebellum at postnatal day 8. Wild-type GCs grown in this culture system display an important developmental property--the ability to extend neurites. However, no inwardly rectifying K+ current is detected in GCs cultured from either wv/wv or +/+ cerebellum under a variety of conditions that activate related channels in other tissues. This suggests that if the Girk2 mutation is responsible for the wv phenotype, it does not act by altering these electrical properties of developing GCs.
A simplified procedure for developing multiplex PCRs.
Authors: Shuber A P , Grondin V J , Klinger K W
Published: June 2007

We have developed a simplified method for multiplex PCR based on the use of chimeric primers. Each primer contains a 3' region complementary to sequence-specific recognition sites and a 5' region made up of an unrelated 20-nucleotide sequence. Identical reaction conditions, cycling times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, efficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additional modification of either the reaction components or annealing temperatures is required. The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.
A physical map of chromosome 2 of Arabidopsis thaliana.
Authors: Zachgo E A , Wang M L , Dewdney J , Bouchez D , Camilleri C , Belmonte S , Huang L , Dolan M , Goodman H M
Published: June 2007

A yeast artificial chromosome (YAC) physical map of chromosome 2 of Arabidopsis thaliana has been constructed by hybridization of 69 DNA markers and 61 YAC end probes to gridded arrays of YAC clones. Thirty-four YACs in four contigs define the chromosome. Complete closure of the map was not attained because some regions of the chromosome were repetitive or were not represented in the YAC library. Based on the sizes of the YACs and their coverage of the chromosome, the length of chromosome 2 is estimated to be at least 18 Mb. These data provide the means for immediately identifying the YACs containing a genetic locus mapped on Arabidopsis chromosome 2.
An integrated YAC map of the human X chromosome.
Authors: Roest Crollius H , Ross M T , Grigoriev A , Knights C J , Holloway E , Misfud J , Li K , Playford M , Gregory S G , Humphray S J , Coffey A J , See C G , Marsh S , Vatcheva R , Kumlien J , Labella T , Lam V , Rak K H , Todd K , Mott R , Graeser D , Rappold G , Zehetner G , Poustka A , Lehrach H
Published: June 2007

The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males. With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map. We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones. Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources. We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones. These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques. This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.
Fine structure of the human galactokinase GALK1 gene.
Authors: Bergsma D J , Ai Y , Skach W R , Nesburn K , Anoia E , Van Horn S , Stambolian D
Published: June 2007

Defects in the human GALK1 gene result in galactokinase deficiency and cataract formation. We have isolated this gene and established its structural organization. The gene contains 8 exons and spans approximately 7.3 kb of genomic DNA. The GALK1 promoter was localized and found to have many features in common with other housekeeping genes, including high GC content, several copies of the binding site for the Sp1 transcription factor, and the absence of TATA-box and CCAAT-box motifs typically present in eukaryotic Pol II promoters. Analysis by 5'-RACE PCR indicates that the GALK1 mRNA is heterogeneous at the 5' terminus, with transcription sites occurring at many locations between 21 and 61 bp upstream of the ATG start site of the coding region. In vitro translation experiments of the GALK1 cDNA indicate that the protein is cytosolic and not associated with the endoplasmic reticulum membrane.
Isolation and characterization of the mouse cystatin B gene.
Authors: Pennacchio L A , Myers R M
Published: June 2007

The cystatins make up a large superfamily of proteins that inhibit cysteine proteases. Recently, we showed that loss-of-function mutations in the human cystatin B gene are responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1). However, despite the known role of cystatin B in cysteine protease inhibition, it is not clear why decreased levels of this protein cause EPM1. To provide new insights into the biochemical and pathological mechanisms of EPM1, we are working toward developing an animal model for this disease. Here we present the mouse cystatin B nucleotide and amino acid sequence. We show that the mouse gene spans a 3-kb genomic region and contains 3 exons and 2 introns, identical to the structure of both the rat and human cystatin B genes. The amino acid sequence identity of the protein is 86%, 79%, and 71% to that of the rat, human, and bovine cystatin B proteins, respectively. In addition, we show that the mouse cystatin B gene is expressed in many tissues, similar to results observed previously in humans. Finally, we report the mapping of the mouse cystatin B gene (Stfb) to chromosome 10, further extending the synteny between this region of the mouse chromosome and human chromosome 21q22.3.
The marine bacterium Pseudoalteromonas haloplanktis has a complex genome structure composed of two separate genetic units.
Authors: Lanoil B D , Ciuffetti L M , Giovannoni S J
Published: June 2007

The genome size of Pseudoalteromonas haloplanktis, a ubiquitous and easily cultured marine bacterium, was measured as a step toward estimating the genome complexity of marine bacterioplankton. To determine total genome size, we digested P. haloplanktis DNA with the restriction endonucleases Notl and Sfil, separated the fragments using pulsed-field gel electrophoresis (PFGE), and summed the sizes of the fragments. The P. haloplanktis genome was 3512 +/- 112 kb by Notl digestion and 3468 +/- 54.1 kb by Sfil digestion. P. haloplanktis is also shown to have a complex genome structure, composed of two large replicons of approximately 2700 and 800 kb. Three pieces of evidence support this conclusion: (1) Two separate bands are always seen in PFGE of undigested P. haloplanktis DNA; (2) restriction digests of the larger band are missing a band of approximately 650 kb compared with restriction digests of total genomic DNA; and (3) a 16S rDNA probe hybridized to the larger replicon but not to the smaller. To our knowledge, P. haloplanktis is the first marine bacterium shown to have a complex genome structure.
Dispersion of human Y chromosome haplotypes based on five microsatellites in global populations.
Authors: Deka R , Jin L , Shriver M D , Yu L M , Saha N , Barrantes R , Chakraborty R , Ferrell R E
Published: June 2007

We have analyzed five microsatellite loci from the nonrecombining portion of the human Y chromosome in 15 diverse human populations to evaluate their usefulness in the reconstruction of human evolution and early male migrations. The results show that, in general, most populations have the same set of the most frequent alleles at these loci. Hypothetical ancestral haplotypes, reconstructed on the basis of these alleles and their close derivatives, are shared by multiple populations across racial and geographical boundaries. A network of the observed haplotypes is characterized by a lack of clustering of geographically proximal populations. In spite of this, few distinct clusters of closely related populations emerged in the network, which are associated with population-specific alleles. A tree based on allele frequencies also shows similar results. Lack of haplotypic structure associated with the presumed ancestral haplotypes consisting of individuals from almost all populations indicate a recent common ancestry and/or extensive male migration during human evolutionary history. The convergent nature of microsatellite mutation confounds population relationships. Optimum resolution of Y chromosome evolution will require the use of additional microsatellite loci and diallelic genetic markers with lower mutation rates.
Isolation and genomic structure of a human homolog of the yeast periodic tryptophan protein 2 (PWP2) gene mapping to 21q22.3.
Authors: Lafrenière R G , Rochefort D L , Chrétien N , Neville C E , Korneluk R G , Zuo L , Wei Y , Lichter J , Rouleau G A
Published: June 2007

As part of efforts to identify candidate genes for disease mapping to the 21q22.3 region, we have assembled a 770-kb cosmid and BAC contig containing eight tightly linked markers. These cosmids and BACs were restriction mapped using eight rare cutting enzymes, with the goal of identifying CpG-rich islands. One such island was identified by the clustering of NotI, EagI, SstII, and BssHII sites, and corresponded to the NotI linking clone LJ104 described previously. A 7.6-kb HindIII fragment containing this CpG-rich island was subcloned and partially sequenced. A homology search using the sequence obtained from either side of the NotI site identified an expressed sequence tag with homology to the yeast periodic tryptophan protein 2 (PWP2). Several cDNAs corresponding to the human PWP2 gene were identified and partially sequenced. Northern blot analysis revealed a 3.3-kb transcript that was well expressed in all tissues tested. A cDNA consensus of 3157 bp was obtained, and an open reading frame potentially encoding 919 amino acid residues was identified. The predicted protein shows 42% identity and 57% similarity at the amino acid level to the yeast PWP2 protein, which is a member of the WD-repeat containing superfamily, and potentially encodes a G-protein beta subunit. The PWP2 gene is split into 21 exons, ranging in size from 53 to 516 bp, and spans an estimated 25 kb. The gene is transcribed in a 21cen-->21qter direction, with its 5' end mapping approximately 195 kb proximal to the 5' end of the phosphofructokinase-liver isoform gene. Four single base-pair polymorphisms were identified using single-stranded conformation polymorphism analysis. Possible functions of the protein based on homology to other members of the WD-repeat-containing family are discussed.
The Xmrk oncogene promoter is derived from a novel amplified locus of unusual organization.
Authors: Förnzler D , Altschmied J , Nanda I , Kolb R , Baudler M , Schmid M , Schartl M
Published: June 2007

Hereditary melanoma in Xiphophorus hybrids is caused by the receptor tyrosine kinase Xmrk. Tumor formation is initiated by overexpression of the Xmrk gene, apparently because of insufficient transcriptional control in the melanocytic lineage of hybrid fish. The oncogenic Xmrk resulted from gene duplication and nonhomologous recombination of the corresponding Xmrk proto-oncogene during evolution. By this event Xmrk was translocated downstream of the promoter of another gene, D (for Donor). This raised the question whether both the Xmrk oncogene and D share similar transcriptional control elements. Studies on the genomic organization of D showed that this gene is amplified in the Xiphophorus genome, presumably with all copies clustered on a single chromosome. Surprisingly, at least two completely different, tightly linked genes are included in the amplified segment. We find a ubiquitously expressed zinc finger gene of the krüppel type, followed by a previously unknown gene, which was the partner of the Xmrk proto-oncogene in the recombination generating the Xmrk oncogene. The nucleotide sequence predicts a gene product with very high amino acid similarity to a hypothetical Caenorhabditis elegans protein. The expression pattern is unrelated to that of the Xmrk oncogene suggesting that despite extended sequence homology a new type of promoter was created by this rearrangement.
Methylation analysis of a marsupial X-linked CpG island by bisulfite genomic sequencing.
Authors: Loebel D A , Johnston P G
Published: June 2007

Paternal X chromosome inactivation occurs in rodent extraembryonic membranes and in all tissues of marsupials. Methylation of CpG islands occurs on the inactive X in eutherians and is considered to be a stabilizing mechanism. The only previous study of a marsupial X-linked CpG island was of the G6PD gene of the Virginia opossum, in which the paternally derived allele is not completely repressed. We have cloned the 5' end of the G6PD gene from an Australian marsupial, the common wallaroo, and sequenced the associated CpG island. The paternally derived G6PD allele is completely repressed in tissues of this species. Methylation analysis using HpaII and Cfol restriction enzymes and bisulfite genomic sequencing of 47 CpG dinucleotides in a 613-bp region reveals hypomethylation of male and female DNA from tissues, cultured fibroblasts (in which the paternal allele is partially expressed) and sperm. This suggests that methylation of CpG islands is not required for maintenance of X inactivation in marsupials even where repression of the paternal allele is complete.
Distribution and evolution of CTG repeats at the myotonin protein kinase gene in human populations.
Authors: Deka R , Majumder P P , Shriver M D , Stivers D N , Zhong Y , Yu L M , Barrantes R , Yin S J , Miki T , Hundrieser J , Bunker C H , McGarvey S T , Sakallah S , Ferrell R E , Chakraborty R
Published: June 2007

We have analyzed the CTG repeat length and the neighboring Alu insertion/deletion (+/-) polymorphism in DNA samples from 16 ethnically and geographically diverse human populations to understand the evolutionary dynamics of the myotonic dystrophy-associated CTG repeat. Our results show that the CTG repeat length is variable in human populations. Although the (CTG)5 repeat is the most common allele in the majority of populations, this allele is absent among Costa Ricans and New Guinea highlanders. We have detected a (CTG)4 repeat allele, the smallest CTG known allele, in an American Samoan individual. (CTG) > or = 19 alleles are the most frequent in Europeans followed by the populations of Asian origin and are absent or rare in Africans. To understand the evolution of CTG repeats, we have used haplotype data from the CTG repeat and Alu(+/-) locus. Our results are consistent with previous studies, which show that among individuals of Caucasian and Japanese origin, the association of the Alu(+) allele with CTG repeats of 5 and > or = 19 is complete, whereas the Alu(-) allele is associated with (CTG)11-16 repeats. However, these associations are not exclusive in non-Caucasian populations. Most significantly, we have detected the (CTG)5 repeat allele on an Alu(-) background in several populations including Native Africans. As no (CTG)5 repeat allele on an Alu(-) background was observed thus far, it was proposed that the Alu(-) allele arose on a (CTG)11-13 background. Our data now suggest that the most parsimonious evolutionary model is (1) (CTG)5-Alu(+) is the ancestral haplotype; (2) (CTG)5-Alu(-) arose from a (CTG)5-Alu(+) chromosome later in evolution; and (3) expansion of CTG alleles occurred from (CTG)5 alleles on both Alu(+) and Alu(-) backgrounds.
Ea recombinational hot spot in the mouse major histocompatibility complex maps to the fourth intron of the Ea gene.
Authors: Khambata S , Mody J , Modzelewski A , Heine D , Passmore H C
Published: June 2007

The majority of recombination events detected within the mouse major histocompatibility complex (MHC) fall into regions of limited physical distance known as hot spots of meiotic recombination. The hot spot associated with the Ea gene appears to be active only in the presence of the p allele carried by the intra-MHC recombinant strain BIO.F(13R). To study the frequency, regulation, and haplotype specificity of recombination at the Ea hot spot, progeny from three different backcrosses involving BIO.F(13R) were screened for recombination events across the MHC using DNA microsatellite markers. Screening of a total of 750 backcross progeny permitted the identification of seven recombinants within the Ea gene. Using restriction site polymorphisms, and sequence-based nucleotide polymorphisms, the recombination breakpoints in all seven Ea recombinants were mapped to two adjacent segments of 71 bp and 346 bp in intron 4 of the Ea gene.
Homoplasy for size at microsatellite loci in humans and chimpanzees.
Authors: Garza J C , Freimer N B
Published: June 2007

Homoplasy (convergence in the size of different alleles) at microsatellite loci was examined by sequencing multiple alleles of two compound microsatellites and single copies of alleles of the same size at two other compound loci in both chimpanzees and humans. At one of the two loci for which multiple alleles were sequenced, extensive homoplasy for size was uncovered both within and between species. At the three loci for which alleles of the same size were examined in the two species, sequencing demonstrated different internal structures. These results confirm theoretical predictions that a certain fraction of mutations at microsatellite loci should produce alleles that are identical in size but differ by a number of mutations. The sequence data reveal a previously unrecognized class of variation at microsatellites and open up the possibility that DNA sequencing may allow the extraction of more information from these loci, thus increasing their power as variable markers for genetic mapping studies. Conversely, the data also indicate that the assumption that alleles of the same size are identical in sequence, which is implicit in several methods of analysis, is violated in some cases. Therefore, caution should be used when employing microsatellites in phylogenetic and other studies in which the individuals being examined are separated by a great number of generations from a common ancestor.
Mapping the RP10 locus for autosomal dominant retinitis pigmentosa on 7q: refined genetic positioning and localization within a well-defined YAC contig.
Authors: McGuire R E , Jordan S A , Braden V V , Bouffard G G , Humphries P , Green E D , Daiger S P
Published: June 2007

Retinitis pigmentosa is a genetically heterogeneous disease that has autosomal dominant, autosomal recessive and X-linked forms. Autosomal dominant retinitis pigmentosa (adRP) has thus far been associated with eight distinct loci, including the rhodopsin and peripherin/RDS genes as well as unidentified genes on chromosomes 7p, 7q, 8q, 17p, 17q, and 19q. The RP10 locus for adRP on chromosome 7q was first mapped in a Spanish family; later, an unrelated American family was identified that also showed linkage to 7q. By combining the linkage results from both families, we are able to assign the disease gene to a 5-cM interval on 7q. Based on extensive physical mapping of this region, the genetic interval is now fully contained within a approximately 5-Mb segment on a well-defined YAC contig. These studies significantly reduce the size of the RP10 critical region, exclude a number of possible candidate genes, and provide the necessary cloned DNA for the positional cloning of the RP10 gene.
Single nucleotide primer extension: quantitative range, variability, and multiplex analysis.
Authors: Greenwood A D , Burke D T
Published: June 2007

The quantitative measurement of transcription products from homologous alleles at a diploid locus has broad application for the study of mammalian gene expression. Single nucleotide primer extension (SNuPE) analysis is a simple and sensitive method for allelic transcript discrimination requiring only 1 bp difference between alleles. In this study the effective range, experimental variation, and the influences of poly(dT)-primed and gene-specific reverse transcriptions are characterized. The ability to analyze several genes from a single reverse transcription reaction is assessed as well. For the genes examined, the maximum range of detection is reached when the minor transcript represents 1/250 of the major allele. Relatively little error is seen within or between assays and linearity of response is maintained over an approximately thousandfold range.
A comprehensive map of the porcine genome.
Authors: Rohrer G A , Alexander L J , Hu Z , Smith T P , Keele J W , Beattie C W
Published: June 2007

We report the highest density genetic linkage map for a livestock species produced to date. Three published maps for Sus scrofa were merged by genotyping virtually every publicly available microsatellite across a single reference population to yield 1042 linked loci, 536 of which are novel assignments, spanning 2286.2 cM (average interval 2.23 cM) in 19 linkage groups (18 autosomal and X chromosomes, n = 19). Linkage groups were constructed de novo and mapped by locus content to avoid propagation of errors in older genotypes. The physical and genetic maps were integrated with 123 informative loci assigned previously by fluorescence in situ hybridization (FISH). Fourteen linkage groups span the entire length of each chromosome. Coverage of chromosomes 11, 12, 15, and 18 will be evaluated as more markers are physically assigned. Marker-deficient regions were identified only on 11q1.7-qter and 14 cen-q1.2. Recombination rates (cM/Mbp) varied between and within chromosomes. Short chromosomal arms recombined at higher rates than long arms, and recombination was more frequent in telomeric regions than in pericentric regions. The high-resolution comprehensive map has the marker density needed to identify quantitative trait loci (QTL), implement marker-assisted selection or introgression and YAC contig construction or chromosomal microdissection.
Mapping of the OB receptor to 1p in a region of nonconserved gene order from mouse and rat to human.
Authors: Chung W K , Power-Kehoe L , Chua M , Leibel R L
Published: June 2007

As part of an effort to identify informative molecular markers for genetic analysis of human pedigrees segregating for obesity, we have developed a genetic map of human 1p in the region of the OB receptor (OBR), the gene that is defective in murine diabetes (Obrdb) and rat Zucker fatty (Obrfa) mutations located on mid-chromosome 4 and chromosome 5, respectively. OBR was mapped 0.9 cR centromeric to WI-9515 and 2.2 cR telomeric of WI-7249 by radiation hybrid (RH) mapping. Ten yeast artificial chromosomes (YACs) containing OBR were identified, confirming the location of OBR centromeric to WI-9515 and telomeric to WI-7249. Additionally, five P1 artificial chromosomes (PACs) were identified that comprised a contiguous series of overlapping clones spanning the length of OBR. WI-5182 was contained within the two PACs that are 3' of OBR. Using a panel of 68 individuals from a single three-generation family and an additional nuclear family, we have mapped 18 polymorphic markers including phosphoglucomutase 1 (PGM1), which is centromeric to Obrdb / Obrfa, and D1S85, which is telomeric to Obrdb / Obrfa in the mouse and rat. The following composite map integrates these radiation hybrid, genetic, and physical maps: Centromere-@WI-7249-[OBR; WI-5182]-D1S198-[WI-9515; WI-6550; D1S2866]-D1S2825-[WI-3077; D1S2886]-[D1S515; DS1613; PGM1]-[D1S312; D1S473; D1S230; D1S246; D1S203]-D2S1643-[D1S1669; D1S1596;]UNCJ-D1S476- D1S85-D1S220-C8B-GTAT1A7. Unresolvable markers are within brackets. A comparison of gene order on mouse chromosome 4, rat chromosome 5, and human 1p indicates that between rodents and humans, there has been a rearrangement of the gene order in the region surrounding OBR.
BCM Search Launcher--an integrated interface to molecular biology data base search and analysis services available on the World Wide Web.
Authors: Smith R F , Wiese B A , Wojzynski M K , Davison D B , Worley K C
Published: June 2007

The BCM Search Launcher is an integrated set of World Wide Web (WWW) pages that organize molecular biology-related search and analysis services available on the WWW by function, and provide a single point of entry for related searches. The Protein Sequence Search Page, for example, provides a single sequence entry form for submitting sequences to WWW servers that offer remote access to a variety of different protein sequence search tools, including BLAST, FASTA, Smith-Waterman, BEAUTY, PROSITE, and BLOCKS searches. Other Launch pages provide access to (1) nucleic acid sequence searches, (2) multiple and pair-wise sequence alignments, (3) gene feature searches, (4) protein secondary structure prediction, and (5) miscellaneous sequence utilities (e.g., six-frame translation). The BCM Search Launcher also provides a mechanism to extend the utility of other WWW services by adding supplementary hypertext links to results returned by remote servers. For example, links to the NCBI's Entrez data base and to the Sequence Retrieval System (SRS) are added to search results returned by the NCBI's WWW BLAST server. These links provide easy access to auxiliary information, such as Medline abstracts, that can be extremely helpful when analyzing BLAST data base hits. For new or infrequent users of sequence data base search tools, we have preset the default search parameters to provide the most informative first-pass sequence analysis possible. We have also developed a batch client interface for Unix and Macintosh computers that allows multiple input sequences to be searched automatically as a background task, with the results returned as individual HTML documents directly to the user's system. The BCM Search Launcher and batch client are available on the WWW at URL http:@gc.bcm.tmc.edu:8088/search-launcher.html.