Homologies in human and Macasa fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.by E.R. Schmidt     

Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes

Plasmid libraries enriched in sequences from single chromosome types have been constructed for all human chromosomes. This was accomplished by transferring inserts from the Charon 21A phage libraries constructed by the National Laboratory Gene Library Project into Bluescribe plasmids. Insert material freed by complete digestion of the phage libraries with HindIII or EcoRI was cloned into the corresponding sites in Bluescribe plasmids. The sizes of the Bluescribe library inserts determined by gel electrophoresis range from near 0 to approximately 6 kb. Fluorescence in situ hybridization (FISH) with the plasmid libraries showed that all hybridize along both arms of the expected (target) chromosome type with varying intensity. However, the plasmid libraries for chromosomes 1, 4, 9, 11, 16, 18, and 20 hybridize weakly or not at all near the centromeres of the target chromosome types. The libraries for chromosomes 13, 14, 15, 21, and 22 cross-hybridize near the centromeres of all members of this group and hybridize weakly to the short arms of the target chromosomes. FISH with each library allows specific staining of the target chromosome type in metaphase spreads. The signals resulting from FISH with libraries for chromosomes 1, 4, 8, 9, 13, 14, 17, 18, 21, and Y are sufficiently intense to permit analysis in interphase nuclei. Examples of the use of these libraries for translocation detection, marker chromosome characterization, and interphase aneuploidy analysis are presented.     

Characterization and mapping of microdissected genomic clones from the adenomatous polyposis coli (APC) region.

The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2-21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6-8 Mb from APC-affected individuals around chromosome bands 5q21-22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

Summary Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.     

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

Summary The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.     

Characterization of Two Chromosome 12 Cosmid Libraries and Development of STSs from Cosmids Mapped by FISH

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LLt2NC01 and LLt2NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.     

利用染色体涂染方法建立人和白眉长臂猿(Hylobates hoolock)的比较染色体图谱

除人Y染色体外,本文采用生物素标记的人全部整条染色体特异探针与白眉长臂猿(Hylobates hoolock)有丝分裂中期分裂相进行染色体原位杂交即染色体涂染法以研究人和白眉长臂猿染色体之间的同源性。在白眉长臂猿18对常染色体上检测出了与人22对常染色体同源的59对染色体片段,确定了人和白眉长臂猿之间的精度较高的染色体连锁群。结果表明:自人与白眉长臂猿的祖先分歧以来,大量的染色体间重排(至少发生了39次易位)和染色体内的重排导致了二者核型的差异。根据杂交结果绘制了首份人和白眉长臂猿比较染色体图谱,并结合已有的人和白掌长臂猿(Hylobates lar)(2n=44)和合趾长臂猿(Hylobates syndactylus)(2n=50)的比较染色体图谱对长臂猿属的染色体进化作了初步的探讨。     

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.     

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.     

Chromosomal localization of Sau3A repetitive DNA revealed by in situ hybridization

The Sau3A DNA family consists of unique alphoid human repetitive DNA which is prone to be excised from the chromosomes and exhibits restriction fragment length polymorphism. We studied the chromosomal localization of the DNA by in situ hybridization using cultured normal human lymphocytes. Under standard hybridization conditions, the sequence hybridized with the centromeric regions of chromosomes 1, 2, 4, 11, 15, 17, 18, 19 and X, but under high stringency hybridization conditions, it hybridized with the centromeric regions of chromosomes 1, 17 and X, and particularly chromosome 11. Based on these results, we discuss the evolutionary relationship among the sequences of the Sau3A DNA family.     

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.     

Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes

The analysis of a recombinant human Y-enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with one single copy or slightly repeated Y-specific DNA sequence, Y-specific repeated sequences of various restriction fragment lengths, Y-chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, Y-specific DNA sequences in addition to multiple X and/or autosomal sequences, or Y-specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46,XY patients with gonadal dysgenesis and small deletions of the Y short arm.     

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.     

Chromosomal in situ hybridization using yeast artificial chromosomes

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.