Biomedical subjects
Publications and source records attributed to D K Green.
Telomeric DNA in the skin cells of 21 human subjects aged between 0 and 92 years was quantified by determining the length of the telomeric smear and the relative amount of TTAGGG repeat sequences. Both telomere length and quantity of telomeric repeat sequences were found to decrease significantly with age. Telomere loss has previously been postulated to be a caused of cell senescence.
We have hypothesized that end-to-end chromosome fusions observed in some tumours could play a part in genetic instability associated with tumorigenesis and that fusion may result from the loss of the long stretches of G-rich repeats found at the ends of all linear chromosomes. We therefore asked whether there is telomere loss or reduction in common tumours. Here we show that in most of the colorectal carcinomas that we analysed, there is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient. We speculate on the consequences of this loss for tumorigenesis. We also show that the telomere arrays are much smaller in colonic mucosa and blood than in fetal tissue and sperm, and that there is a reduction in average telomere length with age in blood and colon mucosa. We propose that the telomerase is inactive in somatic tissues, and that telomere length is an indicator of the number of cell divisions that it has taken to form a particular tissue and possibly to generate tumours.
The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail. The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings. In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated. Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes. The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs.
Analysing and sorting human chromosomes by flow cytometry is a powerful tool in the hands of the molecular biologist. Because of the large number of chromosomes analysed by flow, typically 10(5) for each sample, estimates of the distribution of DNA throughout the chromosome complement of an individual can be made to within three megabase pairs. Rearrangements of DNA in this size range can often be clearly seen and measured by flow cytometry in situations where it was not obvious by traditional cytogenetics. The production of enriched samples of a particular chromosome by flow sorting and the subsequent construction of DNA libraries has played an important part in mapping genes to particular hereditary diseases. Techniques now exist which allow the hybridization of a few thousand sorted chromosomes with characterized or uncharacterized DNA probes to give relatively quick answers about specific chromosome genes. The process of obtaining a sorted chromosome sample from a growing population of peripheral blood lymphocytes or lymphoblastoid cells is compared with other methods of achieving similar results in molecular biological terms. Advances in flow cytometric techniques, which include slit-scanning and hybridization of DNA probes in suspension, are likely to improve the enrichment quality of specific sorted human chromosomes.
A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.
Flow cytometry was used as an additional measurement to monitor 59 untreated patients with bladder cancer. The findings indicate that tumours belonging to the same stage and grade can demonstrate different flow cytometric profiles. Using this technique, together with routine histological assessment, we were able to subclassify bladder tumours in respect of their progress. Flow cytometric profiles are recommended for additional information about the aggressiveness of the tumour and the potential response to treatment.
We have developed a large format digital scanner for several applications in nucleic acid analysis. Here we describe the scanning of autoradiographs of DNA sequencing gels and a set of programs for reading the base sequence. The programs correct distortions in the gel, recognize bands by their characteristic shape and assign bases to bands by weighting band position and intensity. The sequence read in this way is as accurate as that read by an expert.
Flow cytometry (FCM) was used to monitor 24 patients receiving intravesical mitomycin C (MMC) therapy. The patients were selected for MMC therapy because of multiple low stage bladder tumours which had not been cleared by previous endoscopic management. The response to treatment was monitored with cystoscopy, cytology, mucosal biopsies and flow cytometry. Follow-up was for a minimum of 12 months and 19 patients (79%) remained free of tumour. Four patients developed recurrences 6 months after therapy (16.5%) and one showed no response. There were no partial responders. The flow patterns of the four patients who developed recurrence showed an increased RNA/DNA ratio; this was detected after the third MMC instillation and remained throughout the follow-up period. The patients who did not develop recurrence demonstrated either a 1:1 or decreased RNA/DNA ratio. The increased RNA/DNA ratio appears to precede cystoscopic, urine cytology or biopsy evidence of recurrence. If this is confirmed in a larger series it could be used to select patients at high risk for recurrence who would be suitable for alternative therapy.
It is demonstrated that there are a number of advantages in using a mechanised microscope for scoring a large number of metaphase cells from human blood lymphocyte preparations. Following the development of an automatic metaphase spread finding machine based upon a large motorised microscope and a synchronous closed circuit television camera and flashing light source, a much smaller machine which is more appropriate to the cytogenetics laboratory, but with a similar metaphase finding performance has been constructed. The new machine which consists of a Cambridge Instruments 1 micron stepping microscope stage, a linear diode array scanner and a computer is described in detail. Metaphase finding performance figures for various orcein stained human blood lymphocyte preparations are given.
An automatic focusing technique for a computer-controlled optical microscope is described, which is based on an algorithm originally proposed by Mendelsohn and Mayall. The procedure is capable of attaining a focusing accuracy of less than 1/20 micron in the slide-objective distance. The dependence of focusing accuracy and time to focus on the scanner signal-to-noise ratio, and the change in the focus position with time, are discussed.
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