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W R McCombie

Publications and source records attributed to W R McCombie.

At least 19 recordsLinked to original sources
PubMed2005

EST analysis in Ginkgo biloba: an assessment of conserved developmental regulators and gymnosperm specific genes.

BACKGROUND: Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba. RESULTS: RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants--many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads. CONCLUSION: Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms.

Contig Mapping↗
PubMed2003

Evolution and microsynteny of the apyrase gene family in three legume genomes.

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes--and possibly, the acquisition of new functions for nodulation--apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus, Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatulaand L. japonicus), and a genome from an economically important legume species ( G. max).

Apyrase↗
PubMed2001

Identification of mesoderm development (mesd) candidate genes by comparative mapping and genome sequence analysis.

The proximal albino deletions identify several functional regions on mouse Chromosome 7 critical for differentiation of mesoderm (mesd), development of the hypothalamus neuroendocrine lineage (nelg), and function of the liver (hsdr1). Using comparative mapping and genomic sequence analysis, we have identified four novel genes and Il16 in the mesd deletion interval. Two of the novel genes, mesdc1 and mesdc2, are located within the mesd critical region defined by BAC transgenic rescue. We have investigated the fetal role of genes located outside the mesd critical region using BAC transgenic complementation of the mesd early embryonic lethality. Using human radiation hybrid mapping and BAC contig construction, we have identified a conserved region of human chromosome 15 homologous to the mesd, nelg, and hsdr1 functional regions. Three human diseases cosegregate with microsatellite markers used in construction of the human BAC/YAC physical map, including autosomal dominant nocturnal frontal lobe epilepsy (ENFL2; also known as ADNFLE), a syndrome of mental retardation, spasticity, and tapetoretinal degeneration (MRST); and a pyogenic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA).

Abnormalities, Multiple↗
PubMed2000

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

Animals↗
PubMed1999

Genetic definition and sequence analysis of Arabidopsis centromeres.

High-precision genetic mapping was used to define the regions that contain centromere functions on each natural chromosome in Arabidopsis thaliana. These regions exhibited dramatic recombinational repression and contained complex DNA surrounding large arrays of 180-base pair repeats. Unexpectedly, the DNA within the centromeres was not merely structural but also encoded several expressed genes. The regions flanking the centromeres were densely populated by repetitive elements yet experienced normal levels of recombination. The genetically defined centromeres were well conserved among Arabidopsis ecotypes but displayed limited sequence homology between different chromosomes, excluding repetitive DNA. This investigation provides a platform for dissecting the role of individual sequences in centromeres in higher eukaryotes.

Arabidopsis↗
PubMed1999

The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation.

The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9. Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.

Amino Acid Sequence↗
PubMed1999

zA map for sequence analysis of the Arabidopsis thaliana genome.

Arabidopsis thaliana has emerged as a model system for studies of plant genetics and development, and its genome has been targeted for sequencing by an international consortium (the Arabidopsis Genome Initiative; http://genome-www. stanford.edu/Arabidopsis/agi.html). To support the genome-sequencing effort, we fingerprinted more than 20,000 BACs (ref. 2) from two high-quality publicly available libraries, generating an estimated 17-fold redundant coverage of the genome, and used the fingerprints to nucleate assembly of the data by computer. Subsequent manual revision of the assemblies resulted in the incorporation of 19,661 fingerprinted BACs into 169 ordered sets of overlapping clones ('contigs'), each containing at least 3 clones. These contigs are ideal for parallel selection of BACs for large-scale sequencing and have supported the generation of more than 5.8 Mb of finished genome sequence submitted to GenBank; analysis of the sequence has confirmed the integrity of contigs constructed using this fingerprint data. Placement of contigs onto chromosomes can now be performed, and is being pursued by groups involved in both sequencing and positional cloning studies. To our knowledge, these data provide the first example of whole-genome random BAC fingerprint analysis of a eucaryote, and have provided a model essential to efforts aimed at generating similar databases of fingerprint contigs to support sequencing of other complex genomes, including that of human.

Arabidopsis↗
PubMed1999

Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome.

The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements. It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial. We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems. In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes. The representation of unique maize sequences not found in plant reference genomes is also greatly enriched. This demonstrates that repeats, and not genes, are the primary targets of methylation in maize. The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required.

Cloning, Molecular↗
PubMed1998

Kaleidaseq: a Web-based tool to monitor data flow in a high throughput sequencing facility.

Tracking data flow in high throughput sequencing is important in maintaining a consistent number of successfully sequenced samples, making decisions on scheduling the flow of sequencing steps, resolving problems at various steps and tracking the status of different projects. This is especially critical when the laboratory is handling a multitude of projects. We have built a Web-based data flow tracking package, called Kaleidaseq, which allows us to monitor the flow and quality of sequencing samples through the steps of preparation of library plates, plaque-picking, preparation of templates, conducting sequencing reactions, loading of samples on gels, base-calling the traces, and calculating the quality of the sequenced samples. Kaleidaseq's suite of displays allows for outstanding monitoring of the production sequencing process. The online display of current information that Kaleidaseq provides on both project status and process queues sorted by project enables accurate real-time assessment of the necessary samples that must be processed to complete the project. This information allows the process manager to allocate future resources optimally and schedule tasks according to scientific priorities. Quality of the sequenced samples can be tracked on a daily basis, which allows the sequencing laboratory to maintain a steady performance level and quickly resolve dips in quality. Kaleidaseq has a simple easy-to-use interface that allows access to all major functions and process queues from one Web page. This software package is modular and designed to allow additional processing steps and new monitoring variables to be added and tracked with ease. Access to the underlying relational database is through the Perl DBI interface, which allows for the use of different relational databases. Kaleidaseq is available for free use by the academic community from http://www.cshl.org/kaleidaseq.

Arabidopsis↗
PubMed1997

Molecular cloning and functional expression of a serotonin receptor from Caenorhabditis elegans.

A cDNA encoding a serotonin receptor has been isolated from a Caenorhabditis elegans mixed stage cDNA library. The nematode serotonin receptor, designated 5HT-Ce, was permanently expressed in murine Ltk-cells, where it mediates adenylate cyclase attenuation. Sequence analysis and the pharmacological profiles demonstrate its relatedness not only to Drosophila and Lymnae 5HT receptors but also to mammalian 5HT1a receptors. The 5HT-Ce-gene does not map close to the position of any known serotonergic mutations.

Adenylyl Cyclase Inhibitors↗
PubMed1996

Fluorescence-based sequencing of double-stranded DNA by hexamer string priming.

A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.

Automation↗
PubMed1996

High-quality automated DNA sequencing primed with hexamer strings.

The finishing phase of genome sequencing projects is expensive, in part, because of the cost of de novo synthesis of custom primers and the management burden associated with obtaining and using them for primer walking. One approach to reduce these high costs is the use of a presynthesized library of short oligonucleotides (8-10 bases) rather than long primers. The use of such a library eliminates the need for custom synthesis of oligonucleotides, providing the convenience of priming from any site by combining two to three short oligonucleotides to form a string with the required specificity. The first practical implementation of this strategy presented a robust protocol for using hexamer strings with radioisotopic labelling. Whereas versions of this technique have subsequently been implemented on fluorescent sequencers we felt that there was a need to develop and extensively test a protocol that consistently gave read lengths comparable to dye-terminator sequencing with longer primers. We have developed a new two-cycle fluorescent Sequenase terminator procedure for using hexamer strings. We tested this procedure using a set of 32 different 3 hexamer primer strings, each known to be functional to some degree in radioisotopic sequencing, on single-stranded M13mp18 template and ABI 373 DNA sequencers. The overall success rate of priming with these hexamer primer strings is 97% with the failure of only one string. In this case, the corresponding 18-mer primer also failed to produce usable sequence from M13mp18 template. The average read length from reactions successfully primed with the 31 different hexamer strings was 461 bases with > 99% base-calling accuracy. The current protocol is robust enough to be used in virtually any situation where primer walking on single-stranded templates is used. The success rate and read lengths make it universally applicable to the sequencing of single-stranded templates on automated sequencers. It is also amenable to automation.

Automation↗
PubMed1995

Gene trap tagging of PROLIFERA, an essential MCM2-3-5-like gene in Arabidopsis.

Gene trap transposon mutagenesis can identify essential genes whose functions in later development are obscured by an early lethal phenotype. In higher plants, many genes are required for haploid gametophyte viability, so that the phenotypic effects of their disruption cannot be readily observed in the diploid plant body. The PROLIFERA (PRL) gene, identified by gene trap transposon mutagenesis in Arabidopsis, is required for megaga-metophyte and embryo development. Reporter gene expression patterns revealed that PRL was expressed in dividing cells throughout the plant. PRL is related to the MCM2-3-5 family of yeast genes that are required for the initiation of DNA replication.

Amino Acid Sequence↗
PubMed1994

Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.

Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

Base Sequence↗
PubMed1994

Iatrogenic Creutzfeldt-Jakob disease: an example of the interplay between ancient genes and modern medicine.

We tested DNA from 15 centrally infected cases of iatrogenic Creutzfeldt-Jakob disease (CJD) (dura mater or corneal homografts and stereotactic EEG electrodes), 11 peripherally infected cases (native human growth hormone or gonadotrophin), and 110 control individuals for the presence of mutations in the chromosome 20 amyloid gene. No patient or control had any of the known pathogenic point or insert mutations found in familial disease, but allelic homozygosity at polymorphic codon 129 was present in all but two (92%) of the 26 patients, compared with 54 (50%) of the 110 controls (p < 0.001). Pooled data from all identified and tested cases of iatrogenic disease yielded a worldwide total of 56 patients, of whom all but four were homozygous at codon 129 (p < 0.001). These findings support the thesis that homozygosity at codon 129 enhances susceptibility to iatrogenic infections of both central and peripheral origin, with evident implications for the population of dura mater homograft and pituitary hormone recipients whose lives have been complicated by the possibility of exposure to the infectious agent of CJD.

Amyloid↗
PubMed1994

Automated DNA sequencing using 4-color fluorescent detection of reactions primed with hexamer strings.

The use of strings of contiguous short primers rather than a single long primer has the potential to greatly diminish the cost, time and management efforts associated with sequencing by primer walking. For maximum impact, this chemistry must be adaptable to current fluorescent automated sequencers, which, while allowing automated acquisition of data, have reduced sensitivity when compared with radioactive-based sequencing. The ability to use hexamer strings on Applied Biosystems DNA sequencers has now been demonstrated for single-stranded templates. Procedures are described that allow sequence to be obtained up to 400 bases from the priming site. Signal strength is sufficient in this region to allow single base resolution and highly accurate automatic base calling to be performed by the sequencer. While these conditions can no doubt be further optimized, these results show the feasibility of inexpensive primer walking using hexamer string primers on currently available commercial DNA sequencers. This should have a wide range of applications from genome sequencing projects to the sequencing of cDNA clones without the necessity of creating nested deletions or the necessity of spending inordinate amounts of time and money on oligonucleotide synthesis.

Base Sequence↗
PubMed1992

Creutzfeldt-Jakob disease cosegregates with the codon 178Asn PRNP mutation in families of European origin.

We recently discovered an amino acid-altering heterozygous mutation in codon 178 of the PRNP amyloid precursor gene in patients with familial Creutzfeldt-Jakob disease. This mutation is now shown to be associated with the occurrence of disease in 7 unrelated families of Western European origin, among which a total of 65 members are known to have died from Creutzfeldt-Jakob disease. The mutation was detected in each of 17 tested patients, including at least 1 affected member of each family, and in 16 of 36 of their first-degree relatives, but not in affected families with other mutations, patients with the nonfamilial form of the disease, or 83 healthy control individuals. Linkage analysis in two informative families yielded a lod score of 5.30, which, because no recombinants were found, strongly suggests that codon 178Asn is the actual disease mutation.

Adult↗