Cotransfer of syntenic human genes into mouse cells using isolated metaphase chromosomes or cellular DNA

Summary Chromosome-mediated gene transfer (CMGT) of the human genes for hypoxanthine phosphoribosyl transferase (HPRT) and cytosol thymidine kinase (TK1) into HPRT deficient mouse A9 cells or TK deficient Swiss mouse 3T3TK^– cells was found to occur at frequencies at least one order of magnitude higher than DNA-mediated gene transfer (DMGT). The frequency of CMGT into 3T3TK^– cells was reduced by more than an order of magnitude by a posttreatment of the recipient cells with dimethyl sulphoxide (DMSO). After CMGT, expression of the non-selected genes coding for galactokinase (GALK) and acid alpha-glucosidase (GAA), both syntenic with TK1, was observed in a number of transformants. From the pattern of cotransfer, a tentative gene ordering of CENTROMERE-GALK-TK1-GAA on human chromosome 17 was deduced. Chromosome-mediated cotransfer of X-linked human phosphoglycerate kinase (PGK) with HPRT was observed in two out of 33 A9 transformants analysed. DNA-mediated cotransfer of a syntenic gene was only observed for GALK, cotransferred with TK1 in two out of 18 TK⁺ transformants of mouse LTK^– cells. Therefore, with murine cells as recipients of human donor genetic material, CMGT results in a higher frequency of transfer and a higher incidence of cotransfer of syntenic genes than DMGT using cellular DNA in the same cell system.     

Selective digestion of mouse metaphase chromosomes

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000xg pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Restriction enzyme banding of mouse metaphase chromosomes

Fixed metaphase chromosomes from mouse strain RIII embryos or A9 cells were treated with a restriction endonuclease, followed by Giemsa staining. Aha I, Hinf I, or Mbo I treatment produced a C-band pattern, and Eco RII or Hae III produced a G-band plus C-band pattern. Ava II and Bst NI each produced a G-band pattern, but on most chromosomes only a small segment of each C-band, adjacent to the centromere, was stained. These tiny residual C-bands may contain a minor satellite located adjacent to the major satellite clusters.     

In situ random primer extension of metaphase chromosomes

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.     

The structural organization of mouse metaphase chromosomes

The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5-GAAAAATGA-3 (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA      

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Structure of DNA in metaphase chromosomes of mouse fibroblasts

We show that N-1 in adenine of chromosomal DNA is methylated by treatment of metaphase chromosomes with dimethylsulphate while this is not the case in chromatin. The data on methylation are consistent with those obtained from the experiments with S1-nuclease treatment of chromatin and chromosomes. This suggests a disarrangement of DNA secondary structure in the metaphase chromosomes.     

DNAse I digestion of fixed chromosomes specifically reveals NORs in man

Chromosomal preparations were digested with different concentrations of DNAse I for various periods of time and then stained with Giemsa. It was found that this endonuclease rapidly modifies the structure of the chromosomes which then lose most of their stainability. However, small chromosomal segments corresponding to the nucleolar organizers (NORs) in man have been consistently observed. Although DNAse I treatment has permitted us to observe that some NORs are stained less intensely than others, comparisons with silver nitrate-stained NORs have shown a strict correspondence, in metaphase as well as in interphase. Chromosomal proteins seem responsible for this staining.     

Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes

Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using ³H-labeled DNA probes.     

Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes

A technique of in situ hybridization on metaphases of chromosomes by a digoxigenin-labeled probe is described. This technique was able to detect single DNA sequences of 2 and 7 kilobases. The results obtained were compared with those of a biotin streptavidin alkaline phosphatase-based detection system. The digoxigenin method was at least as efficient and sensitive as the biotin-streptavidin method.     

In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.     

Fluorescence analysis of late DNA replication in mouse metaphase chromosomes using BUdR and 33258 Hoechst

A late replicating X or Y chromosome can be detected by 33258 Hoechst staining and fluorescence microscopy in a large proportion of female or male mouse embryo cells, respectively, which have been cultured in medium containing 5-bromodeoxyuridine (BUdR) for part of one DNA synthesis period, The observed distribution of late replicating chromosome regions also includes centromeric heterochromatin and some quinacrine positive bands.     

Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei]

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.     

Non-nucleosomal configuration of chromatin in nucleolar organizer regions of metaphase chromosomes in situ

The structure of metaphase chromatin in a human tumor cell line, TG cells, was investigated using thin sections selectively stained for DNA with the Feulgen-like osmium-ammine reaction. The bulk of metaphase chromatin was characterized by the nucleosomal configuration. Some specimens were pretreated by silver staining for selective visualization of acidic proteins of the nucleolar organizer regions. In these specimens, the osmium-amine DNA tracer revealed that the chromatin present at the sites of silver granule localization had a completely extended configuration, and never gave rise to nucleosomal structures.     

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.