Restriction endonucleases (REs) have been widely used to produce banding patterns on chromosomes, but it remains uncertain to what extent the patterns are due to the sequence specificity of the enzymes, and to what extent chromatin structure influences the pattern of digestion. To throw light on this question, we have digested with restriction endonucleases unfixed chromosomes prepared in two different ways (isolated, and whole metaphase cells spread with a cytocentrifuge) and compared the results with those obtained on conventionally fixed chromosomes. Unfixed isolated chromosomes are easily destroyed by REs; after fixation with cold methanol, which produced minimal alteration to the chromatin structure, the chromosomes are resistant to the action of REs, and conventional methanol-acetic acid fixation is required to permit the induction of banding patterns by REs. Unfixed cytocentrifuge preparations, in which the chromosomes are still surrounded by cytoplasm, are much more resistant to the action of REs, and again banding patterns were only induced after methanol-acetic acid fixation. We conclude that the action of restriction endonucleases on chromosomes is strongly influenced by chromatin organisation, and that methanol-acetic acid fixation is required to permit the induction of conventional banding patterns on 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.

Fresh and 6-day-old fixed chromosome spreads, both untreated and treated with various banding techniques and nucleases, were stained using monoclonal antibodies to double-stranded and single-stranded DNA. DNA in fixed chromosome preparations became progressively denatured with ageing. The staining pattern of untreated chromosomes with anti-double-stranded DNA antibodies (which resembles G-banding) was determined by the conformation of the chromosomal DNA.

The restriction endonucleases HpaII and MspI both cleave the nucleotide sequence CCGG, but the action of HpaII is inhibited if the internal cytosine is methylated. HpaII and MspI were used on fixed chromosomes from bone marrow cells of individuals suffering from chronic myelogenous leukemia and healthy individuals. We found that MspI acts with the same efficiency on all chromosome samples, whereas HpaII extracts more DNA from the chromosomes of leukemic individuals than from the chromosomes of nonleukemic individuals. We postulate that demethylation of cytosine in the CpG dinucleotide of leukemic cell DNA accounts for our findings.

Nuclear Cytology in Relation to Development. By F. d'Amato. Pp. viii+283. (Cambridge University: New York, London and Cambridge, 1977.) £15.

DIPLOID spermatozoa have been found in mouse, rabbit, bull (reviewed by Beatty1) and man2,3. In the rabbit they form 0.03% of the total sperm and in the bull 0.1% to 0.17%, but the incidence in man is not known. Because such sperm could well be involved in the formation of triploid embryos, which are almost invariably aborted in man4, knowledge of their frequency would be of interest.

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β‐glucuronidase, N‐acetyl‐β‐glucosaminidase, and non‐specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa, and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.In the basiphil cells of M. edulis, most of the enzymes are localized in a few cytoplasmic granules; non‐specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.