Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries.

The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and cDNA selection techniques for generating a large resource of new expressed landmarks, which contribute to the construction of a chromosome 21 transcript map

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

Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients

Epub ahead of printSickle cell disease (SCD) is a genetic hemoglobinopathy leading to two major clinical manifestations: severe chronic hemolytic anemia and iterative vaso-occlusive crises. SCD is also accompanied by profound muscle microvascular remodeling. The beneficial effects of endurance training on microvasculature are widely known. The aim of this study was to evaluate the effects of an endurance training program on microvasculature of skeletal muscle in SCD patients. A biopsy of the vastus lateralis muscle and submaximal incremental exercise were performed before and after the training period. Of the forty randomized SCD patients, complete data sets from 32 were obtained. The training group (n=15) followed a personalized moderate-intensity endurance training program, while the non-training (n=17) group maintained a normal lifestyle. Training consisted of three 40-minute cycle ergometer exercise sessions per week for 8 weeks. Histological analysis highlighted microvascular benefits in the training SCD patients compared to non-training patients, including increases in capillary density (CD) (P = .003), number of capillaries around a fiber (CAF) (P = .015) and functional exchange surface (LC/PF) (P < .0001). Conversely, no significant between-group difference was found in the morphology of capillaries. Indexes of physical ability also improved in the training patients. The moderate-intensity endurance exercise training program improved the muscle capillary network and partly reversed the microvascular defects commonly observed in skeletal muscle of SCD patients. This trial was registered at www.clinicaltrials.gov as #NCT02571088

An integrated YAC-overlap and 'cosmid-pocket' map of the human chromosome 21.

We describe here the construction of an ordered clone map of human chromosome 21, based on the identification of ordered sets of YAC clones covering &gt; 90% of the chromosome, and their use to identify groups of cosmid clones (cosmid pockets) localised to subregions defined by the YAC clone map. This is to our knowledge the highest resolution map of one human chromosome to date, localising 530 YAC clones covering both arms of the chromosome, spanning &gt; 36 Mbp, and localising more than 6300 cosmids to 145 intervals on both arms of the chromosome. The YAC contigs have been formed by hybridising a 6.1 equivalents chromosome 21 enriched YAC collection displayed on arrayed nylon membranes to a series of 115 DNA markers and Alu-PCR products from YACs. Forty eight mega-YACs from the previously published CEPH-Genethon map of sequence tagged sites (STS) have also been included in the contig building experiments. A YAC tiling path was then size-measured and confirmed by gel-fingerprinting. A minimal tiling path of 70 YACs were then used as probes against the 7.5 genome equivalents flow sorted chromosome 21 cosmid library in order to identify the lists of cosmids mapping to alternating shared--non-shared intervals between overlapping YACs ('cosmid pockets'). For approximately 1/5 of the minimal tiling path of YACs, locations and non-chimaerism have been confirmed by fluorescence in situ hybridisation (FISH), and approximately 1/5 of all cosmid pocket assignments have independent, confirmatory marker hybridizations in the ICRF cosmid reference library system. We also demonstrate that 'pockets' contain overlapping sets of cosmids (cosmid contigs). In addition to being an important logical intermediate step between the YAC maps published so far and a future map of completely ordered cosmids, this map provides immediately available low-complexity cosmid material for high resolution FISH mapping of chromosomal aberrations on interphase nuclei, and for rapid positional isolation of transcripts in the highly resolved regions of genetic interest