Human chromosome-specific DNA libraries: construction and purity analysis

We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.     

Construction of fifteen human chromosome-specific DNA libraries from flow-purified chromosomes

We report the construction of 15 human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow sorting and the DNA was extracted and cleaved with HindIII before cloning into the lambda vector Charon 21A. A sensitive miniblot hybridization method was used to monitor the physical and biochemical steps in the cloning procedure. Using this method, we have developed a highly efficient protocol for producing large numbers of recombinant phage from 0.2-1.0 X 10(6) sorted chromosomes. DNA from the following chromosomes was cloned: #4, 6, 8, 9, 11, 13, 14 + 15, 16, 17, 18, 19, 20, 21, 22 and Y. These libraries are available to the scientific research community and will be valuable in the genetic analysis of the human genome.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.     

Mapping of rabbit microsatellite markers using chromosome-specific libraries

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.     

Construction and characterization of band-specific DNA libraries

Summary A universally primed polymerase chain reaction was developed to amplify DNA dissected from GTG-banded human chromosomes. The amplification products are cloned into plasmid vectors, which allow the rapid characterization of recombinant clones. Starting from 20–40 chromosome fragments, several thousand independent clones detecting single-copy sequences can be obtained. Although these libraries comprise only a few percent of the dissected DNA, they provide narrowly spaced anchor clones for the molecular characterization of chromosome bands and the identification of gene sequences. Here we describe the construction and characterization of DNA libraries for the Langer-Giedion syndrome chromosome region (LGCR, 8q23–24.1), Wilms tumor chromosome region 1 (WT1, 11p13), Prader-Willi syndrome/Angelman syndrome chromosome region (PWCR/ANCR, 15q11.2–12), meningioma chromosome region (MGCR, 22q12–13), and fragile X chromosome region (FRAXA, Xq27.3).     

Deletion mapping of human chromosome 5 using chromosome-specific DNA probes.

A complete genomic DNA library was prepared from a Chinese hamster-human cell hybrid that contains human chromosome 5 as its only human DNA. Unique or low-copy DNA fragments, isolated form recombinant bacteriophage that contained human DNA inserts, were regionally mapped on chromosome 5 using Southern blot analysis of genomic DNA from a series of hybrid cell lines that were selected as having deletions of various portions of 5q. The chromosome 5-specific DNA library, together with a genetic selective procedure allowing the isolation of hybrid cell lines with deletions of virtually any portion of 5q, will provide a means to construct very accurate physical and recombinational maps of this human chromosome. This system represents an excellent opportunity to examine very precisely the relationship between physical and genetic distances for many loci along the length of this autosome.     

Development of human chromosome-specific PCR primers for characterization of somatic cell hybrids

The human chromosome complement of somatic cell hybrids must be assessed each time the hybrids are grown in culture. We have developed a panel of human-specific oligonucleotide primers for genes that have been mapped to each of the autosomes and to the X chromosome. These primers enable the human chromosome content of hybrids to be assessed rapidly by PCR. The sequence of the primers is presented together with the appropriate conditions for human DNA-specific amplification for each pair.     

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are ∼90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.     

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries

Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat. Received: 11 February 1998 / Accepted: 7 April 1998     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

Linkage mapping of rat Chromosome 5 markers generated from chromosome-specific libraries

Seventy-six novel microsatellite markers with various simple sequence repeat (SSR) motifs are reported in this paper. They were generated on the basis of non-radioactive library screening procedures from flow-sorted rat Chromosome (Chr) 5-specific DNA, and were mapped in three rat backcross populations. Fifty-four of these markers mapped to Chr 5, while the other 22 mapped to other chromosomes of the rat genome. The marker D3Uwm8 is a new microsatellite marker for the rat syndecan 4 (ryudocan) gene. A genotyping protocol based on agarose gel electrophoresis is also provided in this paper. Received: 17 December 1998 / Accepted: 17 February 1999     

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines.

The aneuploid stocks of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat (T. aestivum L.) have been developed mainly in 'Langdon' (LDN) and 'Chinese Spring' (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.     

Characterization of de novo duplications in eight patients by using fluorescence in situ hybridization with chromosome-specific DNA libraries.

Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries was performed on samples from eight patients with de novo chromosomal duplications. In five cases, the clinical phenotype and/or cytogenetic evaluations suggested a likely origin of the duplicated material. In the remaining three cases, careful examination of the GTG-banding pattern indicated multiple possible origins; hybridization with more than one chromosome-specific library was performed on two of these cases. In all cases, FISH conclusively identified the chromosomal origin of the duplicated material. In addition, the hybridization pattern was useful in quantitatively delineating the duplication in two cases.     

A chimpanzee-derived chromosome-specific alpha satellite DNA sequence conserved between chimpanzee and human

We describe a cloned 2.7 kb alpha satellite sequence, Pan-3, from the pygmy chimpanzee (Pan paniscus) that specifically hybridizes in situ to chromosome 19 in the pygmy chimpanzee and to the homeologous human chromosome, no. 17. Using high stringency conditions of hybridization on Southern blots, this sequence hybridized to DNA from both species of chimpanzee (P. paniscus and P. troglodytes) and from human but not to DNA from gorilla (Gorilla gorilla) or orangutan (Pongo pygmaeus). Partial sequence analysis showed that Pan-3 and a previously described human chromosome 17-specific clone have up to 91% sequence identity. To our knowledge this is the highest sequence similarity reported between alphoid subsets from human and any other primate.by T.C. Hsu     

Isolation and characterization of chromosome-specific DNA sequences from a chromosome arm genomic library of common wheat

Isolation, physical mapping and polymorphism of chromosome-specific DNA sequences in wheat are reported. Following the microdissection of the long arm of chromosome 5B (5BL) of common wheat, its DNA was amplified by degenerate oligonucleotide-primed PCR and directly cloned into plasmid vectors. Characterization of the chromosome arm library showed that ∼55% of the inserts are of low-copy nature. Southern analysis using aneuploid lines of common wheat revealed that five of 11 low-copy inserts analyzed map to chromosome arm 5BL; four of these are 5BL-specific. By deletion mapping, the 5BL-specific sequences were located to sub- chromosome arm regions. Based on the hybridization patterns of three 5BL-specific sequences to DNA from a diverse collection of goat-grass ( Aegilops ) and wheat ( Triticum ) species, it was concluded that these sequences emerged at different times in the course of evolution of this group of plant species.     

Molecular characterization of the human VIP prohormone

Two distinct cell groups contain α-melanocyte-stimulating hormone-like immunoreactivity in rat hypothalamus. Only one group, located in the arcuate nucleus, contains other opiocortin peptide immunoreactivity. Combined immunocytochemistry, radioimmunoassay and high performance liquid chromatography, using two different antisera, were used in an attempt to characterise the immunoreactive material present in each cell group. The results thus obtained from normal rats, using an antiserum against α-melanocyte-stimulating hormone and one against COOH-terminal adrenocorticotropin, were compared with those obtained from rats treated neonatally with monosodium glutamate, which destroys the arcuate nucleus.In animals treated with monosodium glutamate, cells of the arcuate nucleus, staining with both antisera, were reduced in number. Cells containing only α-melanocyte-stimulating hormone-like immunoreactivity in the lateral hypothalamus were unaffected. Peptide levels detected by radioimmunoassay with both antisera were reduced in parallel. Chromatographed extracts showed parallel reductions in α-melanocyte-stimulating hormone-like and COOH-terminal adrenocorticotropin-like immunoreactivities.These results suggest that if the immunostained cells of the lateral hypothalamus contain conventional α-melanocyte-stimulating hormone, it constitutes only a very small proportion of the total hypothalamic concentration. However, the possibilities that the antiserum is crossreacting with a different molecular species, or with a similar compound synthesised by a different pathway cannot be excluded.