S.A. Latt

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.

Many Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients have a cytogenetic deletion of 15q11q13. While AS and PWS share a similar cytogenetic anomaly, they have very different clinical phenotypes. DNAs from 4 AS patients were examined using 5 chromosome 15q11q13-specific cloned DNA segments. With the present level of resolution, the molecular deletions between AS and those previously reported for PWS did not appear to differ. However, in contrast to the paternal inheritance of the deleted chromosome 15 observed in the majority of PWS patients, maternal inheritance of the deleted chromosome 15 was demonstrated in the AS patients by restriction fragment length polymorphisms (RFLPs).

A method that allows the specific cloning of DNA fragments absent from patients homozygous or hemizygous for chromosomal deletions is described. The method involves phenol-accelerated competitive DNA reassociation and subsequent molecular cloning of appropriately reassociated molecules. The deletion DNA sample utilized in the competition was isolated from a patient with a minute interstitial deletion in the short arm of the X chromosome. Sheared DNA isolated from a male child, who was diagnosed as having Duchenne muscular dystrophy, chronic granulomatous disease, and retinitis pigmentosa, was combined in a 200-fold excess with Mbo I-cleaved DNA isolated from a 49, XXXXY human lymphoid cell line, and the mixture was subjected to a phenol-enhanced reassociation technique. Analysis of 81 unique segments derived from cloned reassociated DNA molecules has led to the identification of 4 (5%) human DNA fragments that are absent from the male patient's DNA. The 4 clones were localized, on the basis of hybridization with restriction nuclease-digested genomic DNA from a panel of human and human-rodent hybrid cell lines, into three regions surrounding band 21 of the short arm of the normal human X chromosome. These clones are potential linkage markers for the diseases affecting this boy. Each clone, as well as others obtainable by this approach, may also serve as a starting point in the eventual cloning of these three X-linked-disease loci. Extension of this approach to other loci, including human tumors potentially homozygous for small deletions, should also be possible.

Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. We used monoclonal antibody against the transferrin receptor (TfR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 12 1/2-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations; 0.1-1 ng of fetal DNA was obtained in the 20-ml maternal samples. In 7/19 cases, a 222-bp band of amplified DNA was detected, consistent with the presence of male DNA in the isolated cells; 6/7 of these were confirmed as male pregnancies by karyotyping amniocytes. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, we were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further refinement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.

Absorption, fluroescence and circular dichroism measrements on 33258 Hoechst-deoxyribonucleic acid (DNA) complexes are consistent with the existence of two types of dye-binding interactions. One type, which persists at elevated solution ionic strength, is highly specific for adenine-thymine-rich DNA. Dye bound under this condition exhibits efficient fluorescence and strong optical activity. A less specific, largely electrostatic interaction is associated with less intense fluorescence and weaker optical activity. The fluorescence of 33258 Hoechst and several other bisbenzimidazole dyes is less when bound to poly(deoxyadenylate-5-bromodeoxyuridylate) than when bound to poly(deoxyadenlyate-deoxythymidylate). Quenching of 33258 Hoechst fluorescence can also be used to detect biosynthetic incorporation of 5-bromodeoxyuridine into the DNA of living cells. This property of 33258 Hoechst should allow fluorescence-activated cell and chromosome sorting according to the extent of DNA synthesis, providing a bridge between biochemical and cytologic analyses of processes related to DNA replication.