Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections.

We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.

Catch-linker + PCR labeling: a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes.

A simple and efficient method to generate hapten-labeled DNA fragments from a trace amount of YAC DNA isolated by PFGE is described. After agarase digestion of the gel slice containing the resolved YAC recombinant, the purified DNA is digested with Sau3Al and a compatible CL oligonucleotide duplex ligated on. A probe is generated by PCR amplification using a primer complementary to the CL with a single biotin moiety incorporated at the 5' end. When used as a FISH probe, this material yields mapping results superior to Alu-PCR or whole YAC labeling methods and allows sensitive detection of chimerism.

Schizophrenia-associated chromosome 11q21 translocation: identification of flanking markers and development of chromosome 11q fragment hybrids as cloning and mapping resources.

Genetic linkage, molecular analysis, and in situ hybridization have identified TYR and D11S388 as markers flanking the chromosome 11 breakpoint in a large pedigree where a balanced translocation, t(1;11)(q43;q21), segregates with schizophrenia and related affective disorders. Somatic cell hybrids, separating the two translocation chromosomes from each other and from the normal homologues, have been produced with the aid of immunomagnetic sorting for chromosome 1- and chromosome 11-encoded cell-surface antigens. The genes for two of these antigens map on either side of the 11q breakpoint. Immunomagnetic bead sorting was also used to isolate two stable X-irradiation hybrids for each cell-surface antigen. Each hybrid carries only chromosome 11 fragments. Translocation and X-irradiation hybrids were analyzed, mainly by PCR, for the presence of 19 chromosome 11 and 4 chromosome 1 markers. Ten newly designed primers are reported. The X-irradiation hybrids were also studied cytogenetically, for human DNA content, by in situ Cot1 DNA hybridization and by painting the Alu-PCR products from these four lines back onto normal human metaphases. The generation of the translocation hybrids and of the chromosome 11q fragment hybrids is a necessary preliminary to determining whether a schizophrenia-predisposition gene SCZD2 is encoded at this site.

YAC mapping by FISH using Alu-PCR-generated probes.

Human genomic mapping has been greatly advanced by the independent development of three new methods: large DNA fragment cloning in yeast artificial chromosomes, amplification from complex DNAs of human specific segments by Alu-PCR, and high-resolution localization of complex DNA probes by fluorescent in situ hybridization. We describe here the combination of these three analytical tools for efficient and accurate localization of randomly screened or especially selected human YAC recombinants to chromosome 11. We map a YAC clone encompassing the pepsinogen A (PGA) locus to 11q13.1-11q13.3.

Further evidence consistent with Yqh as an indicator of risk of gonadal blastoma in Y-bearing mosaic Turner syndrome.

An 8-year-old girl with some features of Turner syndrome and karyotype 45X/46XY had developed a bilateral gonadoblastoma in her rudimentary ovaries. Her normal Y chromosome showed the characteristic distal fluorescence, as seen in her father's. Another mosaic, this time 45X/46XidicY, and also with some Turner features had rudimentary ovaries, but no gonadoblastoma had developed at age 14. The nature of her idicY, which showed no fluorescent distal Yq and had one of the centromeres inactivated, was confirmed by in situ hybridisation with a Yp-specific probe. Using primers from a human Yp-specific sequence, we amplified DNA extracted from paraffin-embedded ovarian tissue from both cases, and from a normal testicle and a normal ovary as controls. The finding of the expected Y-derived PCR product in the rudimentary gonads from these mosaic patients indicates the presence of their Y chromosome in both. We discuss the validity of the findings, and the possible role of sequences in or near the fluorescent part of Yq in the origin of gonadoblastoma in Y-bearing mosaic Turner syndrome.

Gene mapping to chromosomes by hybridization in situ.

Since the technique of hybridizing labeled nucleic acid sequences to cytological preparations (hybridization in situ) was first described in 1969 by Pardue and Gall (1), it has undergone considerable refinement. In those early days, the technique was only capable of detecting and locating highly repeated target sequences such as satellite DNAs (2) or the genes for ribosomal DNA (3).

Deleted Yq in the sterile son of a man with a satellited Y chromosome (Yqs).

Disturbed spermatogenesis and azoospermia are reported in a man with a deleted Y chromosome. The anomalous Y chromosome appears in the karyotype as a small metacentric marker. In situ hybridisation using three different Y specific DNA probes shows that deletion at Yq11 has resulted in loss of all distal heterochromatin. The sterility of the patient indicates loss also of the azoospermia factor (AZF) located at the Yq distal euchromatic/heterochromatic interface. Microspread and air dried meiotic preparations show a severe impairment of spermatogenesis but rare cells are seen to be progressing to the late prophase stage. The testicular histology shows most of the seminiferous tubules to be completely hyalinised. The father and a fertile brother of the proband show a satellited Y chromosome (Yqs) in their karyotypes. The case appears to be the first of its kind reported in which a father with a satellited Y chromosome has produced a son carrying a different Y chromosome anomaly. The possible derivation of the one from the other is discussed.

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei.

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.

Sexing whole human pre-embryos by in-situ hybridization with a Y-chromosome specific DNA probe.

We have used DNA-DNA in-situ hybridization with a DNA probe for the human Y-chromosome to distinguish between male and female human pre-embryos. Both biotinylated and tritiated Y-probes worked well on control cell cultures where 100 interphase nuclei were scored. Morphologically normal pre-embryos could be sexed with confidence with the tritiated Y-probe but the biotin results were less reliable (although only a few pre-embryos were analysed in this way). Early cleavage stage pre-embryos had large nuclei with relatively diffuse Y-bodies and were more difficult to score with the biotinylated Y-probe. Morphologically abnormal pre-embryos often had large nuclei with multiple Y-bodies (presumably polyploid nuclei) or small nuclei with no Y-bodies (possibly fragmenting nuclei). In all, 38 cleaving and two non-cleaving pre-embryos were analysed. The incidence of false positive and false negative cells seen after hybridization of tritiated Y-probes to control lymphocyte cultures suggests that it should normally be possible to distinguish morphologically normal male and female pre-embryos with samples of three to six interphase nuclei.

Telomeric repeat from T. thermophila cross hybridizes with human telomeres.

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.

Chromosomal organization of the cytochrome P450-2C gene family in the mouse: a locus associated with constitutive aryl hydrocarbon hydroxylase.

Cytochromes P-450 represent a superfamily of enzymes with a central role in the metabolism of drugs, chemical toxins, and carcinogens. We have used genetic analysis to establish the complexity and catalytic function of a recently identified constitutively expressed murine hepatic cytochrome P-450 encoded by P450-2C. Southern blotting analysis shows that there are at least seven or eight genes within this family in the mouse and rat and that DNA restriction fragment length variants between different mouse inbred strains are observed. Analysis of recombinant inbred strains derived from these parent strains shows (i) these genes are clustered within 1 centimorgan, (ii) this gene family does not correspond to any of the known cytochrome P-450 loci or map near any well-characterized genomic markers, and (iii) this gene family segregates to within 1-2 centimorgans of a locus controlling constitutive aryl hydrocarbon hydroxylase activity in mice. With use of Chinese hamster/mouse somatic cell hybrids, the P450-2C locus was assigned to a region of mouse chromosome 19 that appears to be syntenic with the previously mapped human P450C2C locus on human chromosome 10. By in situ hybridization to mitotic mouse chromosomes, we have localized this region to the tip of chromosome 19. These results are discussed in relation to the physiological roles of this P-450 family in foreign compound metabolism and steroid oxidations.

Human cytochrome P-450 PB-1: a multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10.

The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans.