RESUMO
The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate.
Assuntos
Antineoplásicos/química , Lipossomos/química , Análise em Microsséries , Aminoquinolinas/química , Aminoquinolinas/toxicidade , Antineoplásicos/toxicidade , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Descoberta de Drogas , Células HeLa , Humanos , Imiquimode , Microscopia de Força Atômica , Microscopia de Fluorescência , Nanoestruturas/química , Medicina de Precisão , Taxoides/química , Taxoides/toxicidade , Trietilenomelamina/química , Trietilenomelamina/toxicidadeRESUMO
We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.
Assuntos
Drosophila melanogaster/genética , Sequências de Repetição em Tandem , Cromossomo X , Animais , Sequência de Bases , Passeio de Cromossomo , Elementos de DNA Transponíveis , Eucromatina , Heterocromatina , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Análise de Sequência de DNARESUMO
Chromatin on the inactive X chromosome (Xi) of female mammals is enriched for the histone variant macroH2A that can be detected at interphase as a distinct nuclear structure referred to as a macro chromatin body (MCB). Green fluorescent protein-tagged and Myc epitope-tagged macroH2A readily form an MCB in the nuclei of transfected female, but not male, cells. Using targeted disruptions, we have identified two macrochromatin domains within macroH2A that are independently capable of MCB formation and association with the Xi. Complete removal of the non-histone C-terminal tail does not reduce the efficiency of association of the variant histone domain of macroH2A with the Xi, indicating that the histone portion alone can target the Xi. The non-histone domain by itself is incapable of MCB formation. However, when directed to the nucleosome by fusion to core histone H2A or H2B, the non-histone tail forms an MCB that appears identical to that of the endogenous protein. Mutagenesis of the non-histone portion of macroH2A localized the region required for MCB formation and targeting to the Xi to an approximately 190 amino acid region.
Assuntos
Cromatina/metabolismo , Mecanismo Genético de Compensação de Dose , Histonas/química , Histonas/metabolismo , Cromossomo X/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cromatina/química , Cromatina/genética , Feminino , Histonas/genética , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Aberrações dos Cromossomos Sexuais/genética , Especificidade por Substrato , Transfecção , Cromossomo X/química , Cromossomo X/genéticaRESUMO
MacroH2A1 is an unusual variant of the core histone H2A which is enriched in chromatin on the inactive X chromosome of female mammals. The N-terminal third of the protein shares 65% amino acid identity with core histone H2A, while the remaining two-thirds of the protein are novel, with a small stretch of basic amino acids and a putative leucine zipper motif. We have now cloned a second macroH2A gene, encoding macroH2A2 which shares 80% amino acid identity with macroH2A1. Despite mapping to different chromosomes, the genomic organization of the macroH2A2 and macroH2A1 genes are nearly identical. The leucine zipper motif of macroH2A1 is not conserved in macroH2A2. Like macroH2A1, macroH2A2 forms a Macro Chromatin Body in the nuclei of female cells which is coincident with an X chromosome and co-localizes with macroH2A1. To address the distribution of other histone H2A variants in relation to macroH2A and the inactive X chromosome, we constructed a series of epitope-tagged versions of other histone H2A variants. Like the recently described H2A-Bbd (Barr body-deficient) variant, the histone variant H2A.Z was found to be deficient in chromatin on the inactive X chromosome in a significant proportion of female nuclei. This study provides further information about the nucleosomal composition of chromatin on the inactive X chromosome and indicates that a number of H2A variants are non-randomly distributed on the active and inactive X chromosomes.
Assuntos
Mecanismo Genético de Compensação de Dose , Histonas/genética , Cromossomo X , Sequência de Aminoácidos , Animais , Ciclo Celular , Núcleo Celular/metabolismo , Cromatina/genética , Mapeamento Cromossômico , Clonagem Molecular , Evolução Molecular , Variação Genética , Histonas/biossíntese , Histonas/química , Histonas/imunologia , Humanos , Camundongos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.
Assuntos
Cromatina/genética , Histonas/genética , Cromossomo X , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Etiquetas de Sequências Expressas , Feminino , Variação Genética , Heterocromatina/genética , Histonas/biossíntese , Histonas/química , Humanos , Hibridização in Situ Fluorescente , Neoplasias Renais , Mamíferos , Dados de Sequência Molecular , Família Multigênica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP >> ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/enzimologia , Cromatografia em Gel , Cromatografia por Troca Iônica , DNA Complementar , Humanos , Dados de Sequência Molecular , SolubilidadeAssuntos
Química Encefálica/genética , Cromossomos Humanos Par 9/genética , Disautonomia Familiar/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/química , Primers do DNA/química , Humanos , Células Híbridas , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
A novel human transcript CG-2 (C9ORF5), was isolated from the familial dysautonomia candidate region on 9q31 using a combination of cDNA selection and exon trapping. CG-2 was detected as a relatively abundant 8kb transcript in all adult and fetal tissues with the exception of adult thymus. Genomic analysis of CG-2 identified 18 exons that span more than 110kb. The gene encodes a 911-amino-acid protein with a predicted molecular weight of 101kDa and a hypothetical pI of 9.03. Sequence analysis of CG-2 indicates that it is likely to encode a transmembrane protein. Here, we assess CG-2 as a candidate for familial dysautonomia.
Assuntos
Genes de Helmintos/genética , Genes/genética , Proteínas de Membrana/genética , Adulto , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Caenorhabditis elegans/genética , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 9/genética , Clonagem Molecular , Cricetinae , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Bases de Dados Factuais , Disautonomia Familiar/genética , Etiquetas de Sequências Expressas , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.
Assuntos
Actinas/genética , Cromossomos Humanos Par 9/genética , Disautonomia Familiar/genética , Adulto , Sequência de Aminoácidos , Animais , Northern Blotting , Mapeamento Cromossômico , Cromossomos/genética , Clonagem Molecular , DNA/química , DNA/genética , DNA/isolamento & purificação , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muridae , RNA/genética , RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
A novel Drosophila melanogaster gene UBL3 was characterized and shown to be highly conserved in man and Caenorhabditis elegans (C. elegans). The human and mouse homologues were cloned and sequenced. UBL3 is a ubiquitin-like protein of unknown function with no conserved homologues in yeast. Mapping of the human and mouse UBL3 genes places them within a region of shared gene order between human and mouse chromosomes on human chromosome 13q12-13 and telomeric mouse chromosome 5 (MMU5).
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Drosophila melanogaster/genética , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
We have isolated and characterized a novel PHD finger gene, PHF2, which maps to human Chromosome (Chr) 9q22 close to D9S196. Its mouse homolog was also characterized and mapped to the syntenic region on mouse Chr 13. The predicted human and mouse proteins are 98% identical and contain a PHD finger domain, eight possible nuclear localization signals, two potential PEST sequences, and a novel conserved hydrophobic domain. Northern analysis shows widespread expression of PHF2 in adult tissues, while in situ hybridization on mouse embryos reveals staining in the neural tube and dorsal root ganglia significantly above a ubiquitous low level expression signal. From its expression pattern and its chromosomal localization, PHF2 is a candidate gene for hereditary sensory neuropathy type I, HSN1.
Assuntos
Cromossomos Humanos Par 9 , Proteínas/genética , Adulto , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Proteínas de Homeodomínio , Humanos , Camundongos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Homologia de Sequência de AminoácidosRESUMO
A meeting on chromosome 9 was held on Tuesday, 27 October 1998 in Denver, with 38 participants (see appendix). Since the last meeting several of the positional cloning efforts on chromosome 9q have come to fruition, and the most detailed discussion was on 9p. Dr Ian Dunham from the Sanger Centre explained the strategy to be used for sequencing chromosome 9, and encouraged collaboration in the preparatory mapping. He indicated that some priority could be given to those regions where people in the field had a strong interest and could identify relevant PAC clones. At this short meeting it was clearly not possible to construct a comprehensive map of chromosome 9, and it was decided that efforts should be made to maintain links to sources of information on the chromosome 9 web page (http:@www.gene.ucl.ac.uk/chr9/). The discussions at the meeting are summarized in four sections: 9p, 9cen-q31, 9q32-9q34 and comparative mapping. Many of the posters presented at the meeting were also presented at the ASHG meeting (28-31 October 1998). They are listed here and are published in The American Journal of Human Genetics, vol. 63 (supplement). Abstracts for posters presented only at this meeting are appended to this report.
Assuntos
Cromossomos Humanos Par 9/genética , Animais , Mapeamento Cromossômico , Doenças Genéticas Inatas/genética , Humanos , Camundongos , Repetições de MicrossatélitesRESUMO
The human lymphoid cell activation antigen CD39 is a known E-type apyrase that hydrolyzes extracellular ATP and ADP, a function important in homotypic adhesion, platelet aggregation, and removal by activated lymphocytes of the lytic effect of ATP. The recently identified putative rat homologue of CD39L1 has been shown to have E-type ecto-ATPase activity, by hydrolyzing extracellular ATP. We have characterized three novel CD39-like transcripts, CD39L2, CD39L3, and CD39L4, which share extensive amino acid homology with other nucleotide triphosphatases in vertebrates, invertebrates, and plants, suggesting that these genes also encode proteins with ecto-nucleotidase activity. Isolation and sequencing of full-length cDNA clones for each gene identified putative proteins of 485, 529, and 429 amino acids. The expression pattern of all five human members of the gene family was analyzed. CD39L2, CD39L3, and CD39L4 were mapped on the human genome, and the murine homologues identified with the putative map locations were assigned on the basis of regions of conserved gene order between human and mouse chromosomes. The map location of mcd39l4 places the gene within a region associated with audiogenic seizure susceptibility in mouse. This disorder is characterized by convulsions induced by loud high-frequency sound and has been shown to be associated with increased nucleotide triphosphatase activity.
Assuntos
Adenosina Trifosfatases/genética , Antígenos CD/genética , Família Multigênica/genética , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Animais , Antígenos CD/química , Apirase/genética , Northern Blotting , Mapeamento Cromossômico , DNA Complementar , Drosophila melanogaster/genética , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The human ninjurin gene was isolated from a cDNA library enriched for transcripts from band 9q22. A 1.2-kb message was detected for ninjurin in all human tissues studied. The full-length sequence codes for a putative 152-amino-acid protein with 89% identity to the rat ninjurin protein. The mouse homologue was isolated and showed 98% amino acid identity to the rat protein. Mapping by FISH localized mouse ninjurin to mouse chromosome 13, a region that shows synteny with human chromosome 9q22. Genomic characterization of the human gene revealed four exons covering less than 10 kb. The map position of the human gene is between the genetic markers D9S196 and D9S197 on human chromosome band 9q22. This places the gene within the candidate regions for the degenerative neurological disorder hereditary sensory neuropathy type I and the cancer predisposition syndrome multiple self-healing squamous epitheliomata.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Mapeamento Cromossômico , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Fatores de Crescimento Neural/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Moléculas de Adesão Celular Neuronais/metabolismo , Cromossomos Humanos Par 9 , Clonagem Molecular , DNA Complementar , Marcadores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição GênicaAssuntos
Hidrolases Anidrido Ácido/genética , Mapeamento Cromossômico , Hidrolases Anidrido Ácido/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Nucleosídeo-Trifosfatase , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The human CD39-like-1 gene (CD39L1) was isolated from a selected cDNA library enriched for transcripts from regions of human Chromosome (Chr) 9q. Database searches with sequences of one group of clones from the selected cDNA library showed strong amino acid homology to the lymphoid cell activation antigen CD39, an ecto-apyrase gene from human and mouse. The full-length sequence for CD39L1 identified a putative 472 amino acid protein with greater than 60% identity with the chicken muscle ecto-ATPase protein, as well as homology to a number of other known ecto-ATPases and ecto-apyrases from rat, garden pea, yeast, and Toxoplasma gondii. A high level of amino acid identity suggests that CD39L1 is closely related to the chicken muscle ecto-ATPase. The presence of the human ABC2 gene on an overlapping cosmid and hybridizations to somatic cell mapping panels suggest that CD39L1 maps to human Chr 9q34. A mouse homolog was isolated (showing greater than 78% nucleotide sequence identity) and mapped by FISH to mouse Chr 2, the syntenic region of human 9q34. The genomic structure of CD39L1 reveals 9 exons covering less than 7 kb.
Assuntos
Adenosina Trifosfatases/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase , Northern Blotting , Bovinos , Galinhas , Clonagem Molecular , Cricetinae , Desoxirribonuclease HindIII/genética , Cães , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
FKHL15 was isolated from a cDNA library enriched for transcripts from 9q22. Isolation and sequencing of a 3.5-kb cDNA clone identified a putative 376-amino-acid protein with greater than 80% homology over a 100-amino-acid stretch to the forkhead DNA-binding domain. The FKHL15 gene contains a region rich in alanine residues, frequently associated with transcriptional repression. The forkhead genes are believed to play important roles in development and differentiation in many different organisms and have also been implicated in the development of some tumors. The map position of FKHL15 on 9q22 places the gene within the candidate regions for the cancer predisposition syndrome multiple self-healing squamous epitheliomata and the degenerative neurological disorder hereditary sensory neuropathy type I. This is a region frequently lost in squamous cell cancer.
Assuntos
Cromossomos Humanos Par 9/genética , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma de Células Escamosas/genética , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Deleção de Genes , Expressão Gênica , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosAssuntos
Cromossomos Humanos Par 9 , Proteínas Nucleares/genética , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Xeroderma Pigmentoso/genética , Animais , Linhagem Celular , Mapeamento Cromossômico , Cricetinae , Teste de Complementação Genética , Humanos , Células Híbridas , Camundongos , Xeroderma Pigmentoso/metabolismoRESUMO
Using the technique of amplified restriction fragment polymorphism (AFLP) analysis, and bulked segregant pools from F2 progeny of the cross Lycopersicon esculentum (Cf9) x L. pennellii, approximately 42,000 AFLP loci for tight linkage to the tomato Cf-9 gene for resistance to Cladosporium fulvum have been screened. Analysis of F2 recombinants identified three markers which co-segregated with Cf-9. The Cf-9 gene has recently been isolated by transposon tagging using the maize transposon Dissociation (Ds). Analysis of plasmid clones containing Cf-9 shows that two of these markers are located on opposite sides of the gene separated by 15.5 kbp of intervening DNA. AFLP analysis provides a rapid and efficient technique for detecting large numbers of DNA markers and should expedite plant gene isolation by positional cloning and the construction of high-density molecular linkage maps of plant genomes.