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1.
J Pathol ; 216(3): 307-16, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18773450

ABSTRACT

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.


Subject(s)
DNA, Neoplasm/genetics , Gene Dosage , Neoplasms/genetics , Polymerase Chain Reaction/methods , Carcinoma, Bronchogenic/genetics , DNA Primers/genetics , Gene Amplification , Genetic Markers , Genome, Human , Humans , Lung Neoplasms/genetics , Microdissection , Neoplasms/pathology , Paraffin Embedding , Tissue Fixation
2.
Anim Biotechnol ; 18(4): 287-90, 2007.
Article in English | MEDLINE | ID: mdl-17934902

ABSTRACT

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.


Subject(s)
Cattle/embryology , Cattle/genetics , Expressed Sequence Tags , Fetal Death/genetics , Placenta/physiology , Animals , Base Sequence , Chromosome Mapping , Chromosomes, Mammalian , Female , Fetal Death/pathology , Gene Expression Regulation, Developmental , Molecular Sequence Data , Nuclear Transfer Techniques/veterinary , Placenta/metabolism , Placenta/pathology , Pregnancy
3.
Anim Biotechnol ; 18(1): 55-9, 2007.
Article in English | MEDLINE | ID: mdl-17364444

ABSTRACT

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.


Subject(s)
Cattle/embryology , Cattle/genetics , Expressed Sequence Tags , Placenta/physiology , Animals , Base Sequence , Chromosome Mapping , Female , Fetal Death/genetics , Gene Expression Regulation, Developmental , Hybrid Cells , Molecular Sequence Data , Nuclear Transfer Techniques , Pregnancy
4.
Nature ; 435(7038): 43-57, 2005 May 05.
Article in English | MEDLINE | ID: mdl-15875012

ABSTRACT

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.


Subject(s)
Dictyostelium/genetics , Genome , Genomics , Social Behavior , ATP-Binding Cassette Transporters/genetics , Animals , Base Composition , Cell Adhesion/genetics , Cell Movement/genetics , Centromere/genetics , Conserved Sequence/genetics , DNA Transposable Elements/genetics , DNA, Ribosomal/genetics , Dictyostelium/cytology , Dictyostelium/enzymology , Dictyostelium/metabolism , Eukaryotic Cells/metabolism , Gene Duplication , Gene Transfer, Horizontal/genetics , Humans , Molecular Sequence Data , Phylogeny , Proteome , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , RNA, Transfer/genetics , Repetitive Sequences, Nucleic Acid/genetics , Sequence Analysis, DNA , Signal Transduction/genetics , Telomere/genetics
5.
Anim Biotechnol ; 14(1): 83-6, 2003 May.
Article in English | MEDLINE | ID: mdl-12887182

ABSTRACT

Chromosomal mapping of expressed sequence tags for HSPCB and MYL1 expressed abundantly in the bovine fetus was performed by analyzing bovine/murine somatic cell hybrid DNAs with polymerase chain reaction (PCR) using primers specific for those 3'-untranslated regions. HSPCB and MYL1 were assigned to bovine chromosomes 23 and 2, respectively.


Subject(s)
Chromosomes, Mammalian/genetics , Fetus/metabolism , Gene Expression Regulation, Developmental , HSP90 Heat-Shock Proteins/genetics , Myosin Light Chains/genetics , Physical Chromosome Mapping , Animals , Cattle , Expressed Sequence Tags , Hybrid Cells , Mice
8.
Anim Biotechnol ; 12(2): 137-40, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11808628

ABSTRACT

Chromosomal mapping of the bovine calmodulin 1 and alpha-globin 1 genes was performed by analyzing bovine/murine somatic cell hybrid DNAs with PCR using primers specific for 3'-untranslated regions of those bovine genes. The calmodulin 1 and alpha-globin 1 genes were assigned to bovine chromosomes 25 and 29, respectively. Results from the present study should contribute to improvement in map resolution of bovine chromosomes and increase comparative information available on bovine chromosomes.


Subject(s)
Alpha-Globulins/genetics , Calmodulin/genetics , Cattle/genetics , Chromosome Mapping , Animals , DNA Primers , Female , Polymerase Chain Reaction
9.
Genome Res ; 10(11): 1737-42, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11076859

ABSTRACT

We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes.


Subject(s)
Dictyostelium/genetics , Physical Chromosome Mapping/methods , Animals , Blotting, Southern , Contig Mapping/methods , DNA, Protozoan/analysis , Genetic Markers/genetics , Molecular Sequence Data , Radiation Hybrid Mapping/methods , Replication Origin/genetics
10.
Mamm Genome ; 10(12): 1142-5, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10594237

ABSTRACT

In order to assess the extent of DNA sequence variation in cattle, introns and exons from both the leptin and Amyloid Precursor Protein (APP) genes have been sequenced in a panel of DNAs derived from 22 diverse animals. Direct DNA sequencing of PCR products was used; thus, 44 chromosomes were studied. Polymorphisms were identified by manual scanning of sequence chromatograms and computerized sequence analysis. Twenty Single Nucleotide Polymorphisms (SNPs) were detected in 1788 bp sequenced from the leptin gene, giving a frequency of 1 SNP per 89 bp. Twenty-four SNPs were detected in a 458-bp fragment of the APP gene; 23 of the polymorphisms were contained in a 302-bp intron 16 fragment. This equates to an SNP frequency of 1 per 13 bp for the intron. We can thus conclude that this portion of the bovine APP gene constitutes a hypermutable region. Nucleotide sequence diversity values of 0.019 and 0.0026 were obtained for APP and leptin respectively.


Subject(s)
Amyloid beta-Protein Precursor/genetics , Cattle/genetics , Exons/genetics , Introns/genetics , Leptin/genetics , Polymorphism, Genetic/genetics , Amino Acid Substitution , Animals , Chromosomes/genetics , Gene Frequency , Genetic Variation/genetics , Humans , Mutation/genetics , Sequence Analysis, DNA
11.
Mamm Genome ; 10(7): 706-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10384044

ABSTRACT

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.


Subject(s)
Chromosomes , Genomic Library , Animals , Bacteria/genetics , Base Sequence , Cattle , Chimera , Cloning, Molecular , DNA Primers , Genetic Vectors , Male , Microsatellite Repeats
12.
Anim Genet ; 29(4): 302-6, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9745669

ABSTRACT

A bovine/murine hybrid cell panel consisting of 57 cell lines was typed with 124 markers by PCR. Southern hybridisation and isozyme analysis in order to establish its utility as a resource for genome mapping. All bovine chromosomes, including the sex chromosomes were represented in the panel. Computerised analysis of syntenies indicated that there are no cell lines containing only a single bovine chromosome. The panel was used to map 10 new bovine microsatellite markers, and the MYL6 and CPE genes. This panel is informative for all bovine chromosomes other than the sex-specific region of the X chromosome and can be used in synteny mapping studies. At present, due to the relatively small number of markers typed, the resolution of the panel does not go beyond the chromosomal level.


Subject(s)
Cattle/genetics , Chromosome Mapping/methods , Hybrid Cells/chemistry , Animals , Chromosome Mapping/veterinary , Chromosomes/chemistry , Female , Genetic Markers , Male , Mice , Microsatellite Repeats , Tumor Cells, Cultured
18.
Anim Genet ; 24(4): 257-60, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8239069

ABSTRACT

In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.


Subject(s)
Chromosome Mapping/veterinary , Horses/genetics , Adenosine Deaminase/genetics , Aminopeptidases/genetics , Animals , Blotting, Southern , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Genetic Markers , Hybridomas , Isocitrate Dehydrogenase/genetics , Isoenzymes/genetics , L-Lactate Dehydrogenase/genetics , Mannose-6-Phosphate Isomerase/genetics , Mice , Pentosyltransferases/genetics , Peptide Hydrolases/genetics
19.
Cytometry ; 14(4): 369-76, 1993.
Article in English | MEDLINE | ID: mdl-7685678

ABSTRACT

To identify pig chromosomes in pig-mouse somatic cell hybrids, dual-laser flow karyotypes and GTG-banded metaphase spreads of pig, mouse, and 7 pig-mouse hybrid cell lines were compared. Pig chromosomes no. 1, 2, 5, 6, 10, 11, 13, 14, 16, 18, X and Y were tentatively assigned to individual peaks in the pig flow karyotype on the basis of DNA content vs. relative chromosome length. In the 7 hybrid cell lines, 7 out of 8 peaks distinct from those of the mouse cell line could be correlated with the presence of pig chromosomes no. 5, 9, 10, 11 or 16, 14, 15, and 18, whereas 1 peak appeared to correspond to the presence of 1 middle-size chromosome (3, 4, or 7). Other pig chromosomes present in the hybrids could not be detected with certainty due to superposition with mouse peaks and mouse chromosome rearrangements.


Subject(s)
Chromosomes/ultrastructure , Hybrid Cells/ultrastructure , Karyotyping/methods , Swine/genetics , Animals , Bisbenzimidazole , Chromomycin A3 , Chromosome Banding , DNA/analysis , Female , Flow Cytometry , Male , Mice/genetics , Species Specificity
20.
Anim Genet ; 23(1): 51-8, 1992.
Article in English | MEDLINE | ID: mdl-1349204

ABSTRACT

A panel of bovine-murine hybrid cell lines was analysed for 10 loci, including three (IGF1, IGHG2 and the calcium release channel gene [CRC]) that have previously been mapped in man, but not in cattle. The IGF and CRC genes were indirectly mapped to chromosomes 5 and 18 respectively and the syntenies of the HOX2 and GH genes and of the NP and FOS genes were confirmed. The results also show that the IGHG2 locus, which is linked to NP and FOS on human chromosome 14, is separated from these genes in cattle. By showing synteny of the IGHG2 and MPI loci, the IGHG2 locus has been indirectly mapped to chromosome 21.


Subject(s)
Calcium Channels/chemistry , Cattle/genetics , Chromosome Mapping , Genes , Immunoglobulin gamma-Chains/genetics , Insulin-Like Growth Factor I/genetics , Animals , Chromosomes, Human , Genes, Homeobox , Growth Hormone/genetics , Humans , Hybrid Cells , Mice , Multigene Family , Sheep , Swine
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