Flow sorting and microcloning of maize chromosome 1

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.     

The positions of three restriction fragment length polymorphisms on chromosome 4 relative to known genetic markers

Summary A library of DNA Sequences cloned in lambda phage has been prepared from DNA of chromosomes sorted by cytofluorimetry to give enrichment for chromosome 4. Five sequences have been assigned to chromosome 4 using a panel of hybrid cells, and each has been localised relative to a translocation breakpoint at 4q26. Each of the probes gives a Southern blot pattern which indicates that it does not cross-hybridise with sequences found on other human chromosomes. Three of the probes reveal frequent restriction fragment length polymorphisms (RFLPs) and are useful for linkage analysis.     

Construction and characterization of a DNA library from mouse chromosomes 19 purified by flow cytometry

In spite of the constant development concerning physical mapping of eukaryotic genomes, the mouse chromosome 19 remains poorly characterized. In order to improve the possibilities for studying this chromosome, we have constructed a chromosome-specific EcoRI DNA fragment library from mouse chromosomes 19 sorted by flow cytometry. The resulting library contains about 3 X 10(4) recombinant clones. The identified inserts range in size from about 0.2-10 kb, with a 4 kb average size and with no observable redundancy. The purity of the library has been analyzed by flow-blot. For that purpose, chromosomes from 2 cell lines, 1 with a normal karyotype and 1 with translocated chromosome 19, were sorted on nylon filters and hybridized with 9 clones of the library. Results show that 5 clones out of the 9 clearly originate from sorted chromosomes 19 and 3 and are likely to be derived from its DNA, thus indicating that the library of chromosome 19 is of high purity.     

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.     

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.     

Moderately repeated DNA sequences specific for the short arm of the human Y chromosome are present in XX males and reduced in copy number in an XY female.

Four DNA sequences specific for the Y chromosome were isolated from a recombinant phage library constructed from flow sorted human Y chromosomes. Two of these sequences were moderately repeated and assigned to the short arm of the Y chromosome by in situ hybridization. Both sequences were detected in five out of six [corrected] 46,XX males and were reduced in copy number in one out of two 46,XY gonadal dysgenesis patients tested. The findings suggest close proximity of these Y-specific moderately repeated DNA sequences to a testis determining locus.     

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.     

Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome.

We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.     

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.     

Molecular cloning of a DNA fragment from human chromosome 14(14q11) involved in T-cell malignancies.

To isolate DNA segments specific to chromosome band 14q11, which has been implicated in a number of human T-cell malignancies, a genomic DNA library was prepared from a variant cell subline of the human lymphoblastic KE37 cell line. This subline (KE37-R) bears a t(8;14) (q24;q11) translocation, and the breakpoint on the resulting chromosome 8q+ has been located at the 3' end of the third c-myc exon. Three molecular clones were isolated by screening the library with a c-myc exon 3 probe, and one of them (lambda K40) was analyzed in detail. It contains a 15-kb insert consisting of 4.5 kb of sequence from chromosome 8 (e.g., downstream of c-myc exon 3) and sequences from chromosome 14. The origin of these latter sequences was established by hybridizing DNA from chromosomes sorted by flow cytometry to a lambda K40 subclone containing only chromosome 14 presumptive sequences and by Southern blot analysis of rodent X human somatic hybrid cell DNA with the same probe. No cross-hybridization was found between the lambda K40 clone and a cDNA clone for the alpha chain T-cell receptor gene which is also located in 14q11. A preliminary survey of DNAs from human T-cell malignancies with a probe corresponding to chromosome 14 sequences of lambda K40 clone revealed for some of them restriction patterns different from those of the germ line DNA. The fact that the rearrangement observed in a leukemic patient was not found in DNA from lymphocytes obtained during remission excluded any polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of the purity of seven human chromosome-specific DNA libraries

We have characterized at the molecular level seven chromosome-specific libraries constructed in phage lambda Charon 21A from flow-sorted human chromosomes. The purity of libraries prepared from chromosomes sorted from hamster X human cells was estimated by species-specific hybridization and ranged from 48% to 83% of clones containing human inserts. Among libraries of chromosomes from human cells, mass screenings were made for repetitive sequences and 20 clones from the #18 and #20 libraries were analyzed in detail. Ten to fifteen percent of all clones contain sequences which can be mapped; 80-100% of these derive from the intended chromosome of origin, demonstrating very high purity and a 35 X enrichment of chromosome-specific sequences over a total genomic library. The two libraries contain a high, though dissimilar, percent of repeat-containing clones; the #18 library has 55% repetitive clones and the #20 library 85%. This dissimilarity may be due to a difference in insert size distribution, since the #18 library has smaller inserts than the #20. This could be caused by variation in extent of digestion of insert DNA and/or differences in sequence organization between the two chromosomes. A method more sensitive than conventional plaque-lift screening was used to detect repetitive inserts; in this way nearly all repetitive clones could be eliminated before purification of their DNAs.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Isolation of mouse x-chromosome specific DNA from an x-enriched lambda phage library derived from flow sorted chromosomes

A lambda phage library enriched in X(7) chromosomal material has been constructed from flow sorted chromosomes isolated from mice carrying the Cattanach translocation T(X;7)1Ct. The flow sorted fraction that was cloned contained 40% X(7) chromosomes, so that the resulting lambda phage library should be more than 10-fold enriched for X chromosomal DNA. Approximately 100,000 lambda phage clones were obtained; of these, at least 80% were recombinant. Three quarters of recombinants were positive for mouse repetitive DNA as detected either by phage plaque filter hybridization or by Southern blotting. Recombinant DNA inserts were prepared from some of the remaining nonrepetitive phage fraction. The X-chromosome specificity of cloned DNA inserts was tested by hybridization to DNA from mouse-hamster somatic cell hybrids that had retained all or most of the mouse X as the only mouse chromosome and by comparison of the extent of hybridization to DNA from male and female mice. Out of nine cloned unique sequence segments successfully examined thus far, two were presumably derived from the X. Possession of phage library highly enriched for mouse X DNA should facilitate molecular studies of the control of X chromosome gene expression.     

Human ferritin light chain gene sequences mapped to several sorted chromosomes

Summary The iron storage ferritin light-chain gene exhibits multiple restriction enzyme fragments which have been mapped by analyzing sorted human chromosomes. A dual laser chromosome sorter was used to construct spot-blot filter panels representing 22 chromosome fractions. Hybridization of radiolabeled human ferritin-L gene probe to spot-blot panels revealed the ferritin-L gene on more than one chromosome. Miniaturized restriction enzyme analysis was used to map each of the ferritin-L restriction fragments uniquely to one of three chromosomes. This combination of sorted chromosome analyses provides a rapid method to map homologous DNA sequences located on more than one chromosome.     

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries.

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.     

Purifying human Y chromosomes by flow cytometry and sorting

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.     

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY₁Y₂ in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4’,6-diamidino-2-phenylindole bands of both Y chromosomes. Received: 22 December 1998; in revised form: 22 March 1999 / Accepted: 23 March 1999