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There is evidence to suggest that eukaryotic genomes are subject to frequent insertions and deletions of non-coding DNA. This may lead to a gradual increase or decrease in genome size, or to a dynamic equilibrium in which the overall size remains constant. We argue, however, that there is a bias favouring an accumulation of non-coding DNA in the proximity of genes. Such bias causes a progressive change in genome structure regardless of whether the overall genome size increases, decreases or remains constant. We show that this change may serve as a 'molecular clock', supplementing that provided by nucleotide substitution rates.

In the human immune system, antibodies with high affinities for antigen are created in two stages. A diverse primary repertoire of antibody structures is produced by the combinatorial rearrangement of germline V gene segments and antibodies are selected from this repertoire by binding to the antigen. Their affinities are then improved by somatic hypermutation and further rounds of selection. We have dissected the sequence diversity created at each stage in response to a wide range of antigens. In the primary repertoire, diversity is focused at the centre of the binding site. With somatic hypermutation, diversity spreads to regions at the periphery of the binding site that are highly conserved in the primary repertoire. We propose that evolution has favoured this complementarity as an efficient strategy for searching sequence space and that the germline V gene families evolved to exploit the diversity created by somatic hypermutation.

We have identified and sequenced 14 human immunoglobulin VH segments cloned in a yeast artificial chromosome, and have used a rapid PCR-based technique (HAPPY mapping, 12) to derive the order and approximate distances between them. The sequences mapped comprise thirteen germline VH segments and one rearranged VH3 gene. Comparison of our map with other data suggests the existence of at least two distinct haplotypes, differing in the presence or absence of the consecutive genes DP-78, DP-46 and DP-64, and in the duplication of segments DP-49 and DP-65. Screening of ten individuals confirms the existence of both haplotypes, and indicates that both are common amongst the population.

We have devised a simple method for ordering markers on a chromosome and determining the distances between them. It uses haploid equivalents of DNA and the polymerase chain reaction, hence 'happy mapping'. Our approach is analogous to classical linkage mapping; we replace its two essential elements, chromosome breakage and segregation, by in vitro analogues. DNA from any source is broken randomly by gamma-irradiation or shearing. Markers are then segregated by diluting the resulting fragments to give aliquots containing approximately 1 haploid genome equivalent. Linked markers tend to be found together in an aliquot. After detecting markers using the polymerase chain reaction, map order and distance can be deduced from the frequency with which markers 'co-segregate'. We have mapped 7 markers scattered over 1.24 Mbp using only 140 aliquots. Using the 'whole-genome' chain reaction, we also show how the approach might be used to map thousands of markers scattered throughout the genome. The method is powerful because the frequency of chromosome breakage can be optimized to suit the resolution required.

Long DNA molecules of greater than 10(5) bp (0.1 Mbp) are easily broken by pipetting. Therefore, chromosomal DNA is generally isolated after embedding cells in a protective coat of agarose. The embedded DNA can then be cut into long pieces and fractionated on gels using pulsed fields, but these pieces are again easily broken if the resolved DNA molecules are recovered from the gels. We now describe a novel gel matrix, a 'cellular' gel, that permits the recovery of resolved fragments from gels in a form that enables facile manipulation without shear. This facilitates purification and restriction mapping of fragments of 0.1-1.0 Mbp. We illustrate the utility of the method by mapping chromosome III of baker's yeast, which has a length of approximately 0.36 Mbp. This method should facilitate purification and restriction mapping of yeast artificial chromosomes.

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.

A theoretical approach for linkage mapping the genome of any higher eukaryote is described. It uses the polymerase chain reaction, oligonucleotides of random sequence and single haploid cells. Markers are defined and then the DNA of a single sperm is broken at random (eg by gamma-rays) and physically split into 3 aliquots. Each aliquot is screened for the presence of each marker. Closely-linked markers are more likely to be found in the same aliquot than unlinked markers. The entire process is repeated with further sperm and the frequency that any two markers co-segregate determined. Closely-linked markers co-segregate from most cells; unlinked markers do so rarely. A map can then be constructed from these co-segregation frequencies. A specific application for determining the order and distance between sets of closely-linked and previously-defined markers is also described.