Judith A. Fantes

Structure and Evolution of Human Y Chromosome DNA

Two repeated sequences account for 70% of the DNA of the human Y chromosome. They are located in the heterochromatin of the long arm. These sequences are related to others found on human chimpanzee and gorilla autosomes, and on the human X chromosome but have diverged in a characteristic way from the non Y copies. They have no detectable phenotypic effect when translocated to autosomes. We have cloned DNA from the human Y chromosome using fluorescence activated cell sorting. At least one single copy sequence is present on both the X and Y chromosomes.

Karyotyping and identification of human chromosome polymorphisms by single fluorochrome flow cytometry

SummaryFrequency distributions of fluorescence intensity of ethidium bromide stained human chromosomes from nine phenotypically normal males are cross correlated and autocorrelated following repeated flow cytometric measurements. It is shown that each individual donor produces a fluorescence profile which is both visually and numerically different from those of other individuals in the set. The wide variety of chromosome heteromorphisms which occur to varying degrees for chromosomes 1, 9, 13, 14, 15, 16, 21, 22 and Y give rise to the uniqueness of a given fluorescence profile. Estimates of chromosome heteromorphisms for each individual in the set were made and then compared with parallel results obtained from inspection of Q-banded and C-banded conventional metaphase preparations. Fluorescence profiles identifiable with each individual were also obtained for Hoechst 33258 stained chromosomes.

Radiation Dosimetry Using the Methods of Flow Cytogenetics

The measurement of whole body radiation dose by manually counting the proportion of damaged chromosomes in a sample of cultured peripheral blood lymphocytes requires many hours of skilled technician time. A low dose of approximately 15 rads could only be measured with certainty after 500 metaphase cells had been scored and one technician would spread this task over a period of 2–3 days. Flow cytogenetics offers an alternative scoring technique which is potentially more than an order of magnitude faster and which does not involve technician fatigue. We have studied the effects of in vitro radiation doses ranging from 25–400 rads on human peripheral blood lymphocytes by measuring the distortion of the Hoechst 33258 fluorescence distribution of human chromosomes in flow. The level of low intensity background signals, which increases with increasing radiation dose, correlates numerically with the radiation dose administered and the amount of chromosome damage scored by manual means. A human chromosome centromeric staining technique has been investigated which could lead to a method of recognising dicentric chromosomes in flow and a further enhancement to the radiation dose sensitivity of flow cytogenetics.

Human chromosome analysis and sorting.

Flow cytometry has provided the cytogeneticist with a fast and accurate method of measuring the quantity of DNA in each human chromosome (1). Almost all the chromosomes in the human complement can now be resolved and abnormal chromosomes and aneuploidies (13,21, and X) recognized. A flow karyotype shows a pattern of peaks and troughs that is unique for each individual or cell line because of the variation in heterochromatic regions of the chromosomes (2). When combined with family studies, flow cytometry has been able to resolve homologues differing in DNA content by as little as 1/2000 of the human genome (3,4), less than a metaphase band. In addition, the sorting capabilities of most flow machines have provided a method for the purification of small but useful quantities of individual chromosomes, for example, 2×10(6) average sized human chromosomes are equivalent to 500 ng of DNA. Using recombinant DNA techniques, this material can be used to generate a large number of DNA probes to produce a chromosome-specific library, which can be used for the molecular analysis of genetic disease (5,6). More recently, molecular biologists have experimented with gene mapping by sorting small quantities of individual chromosomes onto filters for spot-blot hybridization with DNA probes (7).

ISOLATION OF ANONYMOUS DNA MARKERS FOR HUMAN-CHROMOSOME 22Q11 FROM A FLOW-SORTED LIBRARY, AND MAPPING USING HYBRIDS FROM PATIENTS WITH DIGEORGE SYNDROME

SummaryDiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.

Human Metaphase Chromosomes

Flow cytometry (Melamed et al., 1979) has provided the cytogeneticist with a fast and accurate method for measuring the quantity of DNA in each human chromosome and, more significantly perhaps, a method for purifying chromosomes that have similar amounts of DNA. Analysis rates are usually in excess of 1000 chromosomes/sec, and following the accumulation of fluorescence intensity data from, say 105 chromosomes, which typically takes less than 2 min, it is claimed that chromosome DNA can be estimated to an accuracy of one band, which is 1/600th of the genome (Langlois et al., 1982). Isolating chromosomes in the same group takes somewhat longer. When metaphase chromosomes are passed through a flow cytometer at a rate of 1000/sec, 106 homologous chromosomes from one group will be accumulated over a period of 6 hr.