Chromosomes for molecular hybridization

Summary Specific recombinant DNA sequences (5S rRNA, B1, albumin) were assigned to flow sorted chromosomes of the Chinese hamster cell line CHV79. For this purpose, a rapid protocol was developed using filterbound chromosomal DNA and probing with various nucleic acids, that allows sequence identification in chromosomes. A flow histogram and a flow karyogram of the CHV79 cell line were established by flow analysis in order to calculate the amount of DNA per CHV79 cell and their chromosomes. Subsequently, metaphase chromosomes or chromosomal groups were fractionated by electronic sorting and a defined number of chromosomes was directly bound to nitrocellulose filters for sequence homology analysis by a dot blot hybridization procedure. This procedure not only allows the assigning of specific DNA sequences to particular chromosomes, it is also applicable to studies of changes in karyotypes, for example translocations of given sequences.Some results shown constituted a Diploma Thesis by G.L. submitted to and accepted by the Department of Biology, University of Kaiserslautern     

Assignment of snRNA gene sequences to the large chromosomes of rat kangaroo and Chinese hamster isolated by flow cytometric sorting

Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). — Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.     

A flow cytometric study of chromosomes from rat kangaroo and chinese hamster cells

Summary Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was optimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10 DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated into 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in the karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing high power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influenced by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.     

Localization of genes on fractionated rat chromosomes by molecular hybridization

Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By cross-hybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunoglobulin genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.     

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.     

A method for nucleic acid hybridization to isolated chromosomes in suspension

Summary A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster x human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting.     

Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization.

The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.     

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.     

Bivariate flow karyotyping of acute myelocytic leukemia in the BNML rat model

Univariate as well as bivariate flow karyotyping has been performed on chromosome suspensions obtained from the Brown Norway myelocytic leukemia (BNML), a rat model for human acute myelocytic leukemia (AML). Flow karyograms were obtained from both the in vivo transplantable parent line and from an in vitro established cell line. Density gradient centrifugation performed on cells arrested in mitosis resulted in an enrichment of mitotic cells. Furthermore, with this procedure leukemic and nonleukemic cells could be separated. Univariate analysis with propididum iodide (PI) as a DNA stain revealed the position of the several tumor-specific marker chromosomes in the in vitro cell line. Estimations of the peak position of the various chromosomes was done by comparing the univariate flow karyogram with a computer-simulated karyogram from the BNML that was derived from the mean length of the individual chromosomes in conventionally prepared metaphase slides. By comparing the bivariate flow karyogram of the in vivo BNML cells with the flow karyogram of normal BN cells, it was clearly demonstrated which peaks are involved in the altered chromosomal pattern of the BNML. No differences were found between the flow karyograms of the in vitro- and the ex vivo-derived chromosome suspensions in this rat leukemia model.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.     

USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.     

Flow cytometric chromosome sorting in plants: The next generation

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.     

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.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.     

The integrity of metaphase chromosomes of Haplopappus gracilis (Nutt.) gray isolated by flow cytometry

Metaphase chromosomes were obtained from cell suspension cultures of Haplopappus gracilis (Nutt.) Gray using a mass isolation procedure, and were subsequently sorted by flow cytometry. The integrity of the sorted chromosomes, isolated at neutral pH, was examined by electron microscopy and DNA analysis.Electron microscopy showed no significant damage in the DNA leaked out of the chromosomes at high pH. Sedimentation of chromosomal DNA in alkaline sucrose gradients showed the presence of high molecular weight single stranded DNA fragments (mean MW 4–20 × 10⁸ daltons).These characteristics indicate preservation of DNA integrity and general chromosome structure during the synchronization of the cell cycle, the preparation of protoplasts and the subsequent chromosome isolation procedures. Implications for the use of such chromosomes for genetic manipulation are discussed.     

Localization of specific repetitive DNA sequences in individual rice chromosomes

This paper describes the characterization and chromosomal distribution of three different rice (Oryza sativa) repetitive DNA sequences. The three sequences were characterized by sequence analysis, which gave 355, 498 and 756 bp for the length of the repeat unit in Os48, OsG3-498 and OsG5-756, respectively. Copy number determination by quantitative DNA slot-blot hybridization analysis showed 4000, 1080 and 920 copies, respectively, per haploid rice genome for the three sequences. In situ DNA hybridization analysis revealed that 95% of the silver grains detected with the Os48 probe were localized to euchromatic ends of seven long arms and one short arm out of the 12 rice chromosomes. For the OsG3-498 repetitive sequence, the majority of silver grains (58%) were also clustered at the same chromosomal ends as that of Os48. The minority (28%) of silver grains were located at heterochromatic short arms and centromeric regions. For the OsG5-756 repetitive sequence, 81% of the silver grains labeled the heterochromatic short arms and regions flanking all of the 12 centromeres. Thus, each of these three repetitive sequences was distributed at specific defined chromosomal locations rather than randomly at many chromosomal locations. The approximate copy number of a given repetitive DNA sequence at any specific chromosomal location was calculated by combining the information from in situ DNA hybridization analysis and the total copy number as determined by DNA slot-blot hybridization.by J. Huberman     

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA

A method that permits the in vitro amplification and cloning of DNA dissected from specific regions of a chromosome and does not require prior knowledge of the DNA sequence is described. DNA from several different chromosomal loci in the Drosophila melanogaster genome has been isolated by this method. Although the procedure was developed to permit the isolation of DNA sequences from serial sections of a single microdissected polytene chromosome, it should be useful for obtaining DNA clones from specific regions of the nonpolytene chromosomes of other organisms as well.