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Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

Twelve different human keratinocyte strains were transformed with recombinant plasmid pSV6-1 which contained an origin defective SV40 genome. When injected into athymic nude mice lines produced either squamous cell carcinomas (SCC) in all animals, SCC in some animals and epidermal cysts in others, or epidermal cysts only in all the animals. The tumourigenic capacity of the lines could be correlated with the chromosomal changes present initially in the transformed cells. Lines which produced SCC in all the animals within a short period of time all showed simultaneous loss of part of chromosomes 3p, 8p and 11p in one homologue. Lines which were not tumourigenic did not show these simultaneously appearing rearrangements. These specific rearrangements are acquired in vitro and the time taken for a recognisable tumour to appear is related to the proportion of such cells in the line. The rearrangement of the same chromosome regions in different tumourigenic cell lines suggests that genes in these regions are important in the development of squamous cell carcinoma, possibly by loss of heterozygosity, at particular loci.

A yeast DNA sequence that behaves as a chromosomal replicator, ars1 (autonomously replicating sequence), has been isolated. On transformation, ars1 allows autonomous replication of all co-linear DNA. The replicator can integrate into other replication units and can function in multimeric form. The 850-base pair ars1 element has no detectable homology to other yeast sequences. Such replicator-containing plasmids can be used for the isolation of DNA sequences in yeast cells as well as for the study of chromosomal DNA replication.

Covalently closed circular (ccc) DNAs were isolated by a technique involving alkaline denaturation from the spiramycin producer Streptomyces ambofaciens KA-1028 and also from spiramycin non-producing strains AF-11 and QN-25; plasmids could not be detected in these strains by a cleared lysate method. It was found that most of the ccc DNA in these strains was present in the chromosomal and membrane fractions. These ccc DNAs had identical mobilities in agarose gel electrophoresis. The size was calculated to be 53.1 x 10(6) daltons from the contour length measurements. The ccc DNA gave one fragment on digestion with Hind III, three fragments with Eco R1, and twenty-eight fragments with Bam H1.

New strucutral data have been obtained from neutron scattering studies of chromatin. The concentration-dependent meridional peak at 10-11 nm comes from the interparticle spacing of a subunit structure. Peaks at 5.5 and 3.7 nm have a different contrast behaviour to those at 11.0 and 3.7 nm showing that histones and DNA have a different spatial arrangement in the subunit. A globular model in which apolar segments of histones from the core surrounded by DNA complexed with the basic segments of histones agrees with the data.