Selection of a human chromosome 21 enriched YAC sub-library using a chromosome-specific composite probe.

The subdivision of total genomic human yeast artificial chromosome (YAC) libraries into specific chromosome clone collections will greatly facilitate the construction of an integrated genetic, physical and transcriptional map of the genome. We report the isolation of 388 YAC clones from a human library with an average insert size of 620 kilobases (kb) by the hybridization of a composite chromosome 21 probe to a high-density array of YAC clones. Roughly half of these clones hybridize to chromosome 21 by fluorescence in situ hybridization. These clones represent a twofold coverage of the chromosome. The technique offers the potential of sub-dividing whole genomic YAC libraries into their chromosomal elements to produce high-resolution tools for genome mapping.

A cosmid contig and high resolution restriction map of the 2 megabase region containing the Huntington's disease gene.

The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome.

A pancreatic cancer-specific expression profile.

We present an approach making use of technology established in the context of the genome project to describe a pancreatic cancer-specific expression profile and to identify new potential disease genes or disease-associated-genes. By use of gridded arrays of pancreatic cancer cDNA libraries and differential hybridizations we show that 4% the gridded cDNA library clones contain sequences preferentially expressed in pancreatic cancer. EST-sequencing of 369 distinct (408 total), differentially expressed sequences identified novel genes (32.5%) or homologs to EST-sequences with unknown function (26.3%). Homologies to known genes allow to determine a pancreatic cancer-specific expression profile, which provides for the first time evidence for complex primary and secondary alterations of gene expression responsible for the development of the phenotype of pancreatic cancer cells. In addition this has led to the identification of novel differentially expressed genes, which represent potential oncogenes or disease-associated markers and may be helpful for the development of therapeutic or diagnostic modalities.

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Rapid restriction mapping of DNA cloned in lambda phage vectors.

A protocol for the rapid restriction mapping of phage lambda clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive lambda DNA termini by hybridisation with [32P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the "ladder" of partial digestion products.

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Relational genome analysis using reference libraries and hybridisation fingerprinting.

The genomes of eukaryotic organisms are studied by an integrated approach based on hybridisation techniques. For this purpose, a reference library system has been set up, with a wide range of clone libraries made accessible to probe hybridisation as high density filter grids. Many different library types made from a variety of organisms can thus be analysed in a highly parallel process; hence, the amount of work per individual clone is minimised. In addition, information produced on one analysis level instantly assists in the characterisation process on another level. Genetic, physical and transcriptional mapping information and partial sequencing data are obtained for the individual library clones and are cross-referenced toward a comprehensive molecular understanding of genome structure and organisation, of encoded functions and their regulation. The order of genomic clones is established by hybridisation fingerprinting procedures. On these physical maps, the location of transcripts is determined. Complementary, partial sequence information is produced from corresponding cDNAs by hybridising short oligonucleotides, which will lead to the identification of regions of sequence conservation and the constitution of a gene inventory. The hybridisation analysis of the cDNA clones, and the genomic clones as well, could potentially be expanded toward a determination of (nearly) the complete sequence. The accumulated data set will provide the means to direct large-scale sequencing of the DNA, or might even make the sequence analysis of large genomic regions a redundant undertaking due to the already collected information.

The Reference Library System--sharing biological material and experimental data.

Cosmid, yeast artificial chromosome (YAC), P1 and complementary DNA (cDNA) libraries are distributed on high-density filters to the scientific community and experimental results are stored in a common object-based database accessible through the Internet.

A computer program package for restriction map analysis and manipulation.

Programs for the calculation, storage and analysis of restriction maps derived from the analysis of partial digestion products from end labelled DNA (1,2,3) and their correlation with digestion - and hybridisation patterns in total digestions and Southern blot experiments are described. These programs allow direct input of gel patterns from partial or complete digestion experiments using a digitizer tablet, calculation of molecular weights and restriction maps, plotting of maps and actual or predicted fragment patterns and automated identification of overlapping cosmids from partial restriction mapping results. Programs are written in PASCAL and have been implemented on a VAX/VMS system, with a HP-7221T plotter and a digitizing tablet.

Fast and sensitive determination of furosine.

Sensitive determination of furosine in acid hydrolysates of foods was achieved by isocratic ion-pair reversed-phase HPLC and direct UV-detection within a run time of 5 minutes and levels lower than 1.5 mg per kg of protein. The formation of furosine during hydrolysis of food samples with hydrochloric acid of varying concentration was studied. Furosine formation increased linearly with acid concentration (4 to 8 mol/L).

Construction and preliminary analysis of the ICRF human P1 library.

P1 clone libraries have now been established as effective complements to cosmid and yeast artificial chromosome libraries in long-range mapping projects. To allow general access to P1 clones, we have constructed human and mouse P1 libraries. Clones have been picked into microtiter plates and used to prepare high-density filter grids, providing an efficient and easy screening system. Filters are being made available to other laboratories through the Reference Library System. In this work, we have developed a reliable protocol for generating P1 clones, based on the use of pulsed-field gel electrophoresis for size selection of DNA. A 1.2x genome coverage human library has been produced using this method. A preliminary analysis of this library is described.

Inactivation kinetics of gamma-glutamyltransferase during the heating of milk.

The inactivation of gamma-glutamyltransferase (GGT, E.C.2.3.2.2) during the heating of milk to between 65.0 degrees C and 76.0 degrees C for periods of 5 s to 1000 s follows a first-order reaction (energy of activation 457 kJ/mol) and can be used to monitor the process of milk pasteurisation. GGT activity in raw milks from individual cows showed only little variation (5.92 +/- 0.59 units). Residual GGT activity in 17 commercial milks ranged between 1% and 20%, indicating a heat treatment at the upper limit of the permitted pasteurisation conditions.

Efficient identification and regional positioning of YAC and cosmid clones to human chromosome 21 by radiation fusion hybrids.

The use of integrated mapping strategies involving bacterial, yeast, and rodent cells as hosts simplifies the construction of maps, which combine long-range order, high resolution, and easy access to the cloned DNA. Radiation-fusion hybrids offer a specially powerful long-range mapping system for human chromosomes. We describe here techniques for establishing a radiation-fusion hybrid map of Chromosome (Chr) 21q and its integration with local information on YAC and cosmid positions.

Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.

We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.

Hybridization fingerprinting of high-density cDNA-library arrays with cDNA pools derived from whole tissues.

As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (> 0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database.

Molecular approaches to genome analysis: a strategy for the construction of ordered overlapping clone libraries.

Here we describe progress on a series of molecular techniques designed to bridge the gap between genetic and molecular distances in mammals. This is an essential step in the molecular cloning of genes defined by mammalian mutations, and in the molecular analysis of large regions of mammalian genomes. We summarize approaches for the physical and molecular analysis of genetic distances and describe the experimental, statistical and computational basis of a new approach to create ordered libraries of overlapping clones from large genomes.

Gridded genomic libraries of different chordate species: a reference library system for basic and comparative genetic studies of chordate genomes.

The use of genomic libraries maintained in arrayed format is becoming a more and more popular tool for the analysis of molecular evolution and comparative molecular development. Being able to use already existing reference libraries considerably reduces the work load, and if results are made publicly available, it will facilitate in silica experiments in the future. Here we describe the construction and preliminary characterization of six cosmid libraries of different chordate species, Ciona intestinalis (Hemichordate), Branchiostoma floridae (Cephalochordate), Lampetra fluviatilis (Cyclostoma), Xiphophorus maculatus, and Danio rerio (Osteichthyes) in Lawrist7 and Fugu rubripes in Lawrist4.