Chinese hamster ovary cells were treated with various physical and chemical mutagens and subject to the bromodeoxyuridine plus visible light procedure for isolation of auxotrophic mutants. more than 200 auxotrophs have been isolated which require exogenous supplement of various nutrilites for growth, such as glycine, adenine, thymidine, inositol, etc. Fifty-five of these have been characterized by complementation tests and were shown to constitute 15 different loci. All these auxotrophs are highly stable and exhibit all-or-none growth response to the respective nutrilites. Enzyme deficiencies have been identified in several of these classes. When these auxotrophs are hybridized with human cells and grown in selective medium, the hybrids lose human chromosomes at a rapid rate and the analysis for synteny of the human genes can be successfully carried out. Moverover, in hybrids formed between an adenine-requiring auxotroph and human cells, a human esterase activator gene has been identified which appears to regulate the expression of esterase gene activities in the Chinese hamster genome. Studies of this kind may lead to the understanding of gene regulation in mammalian cells.
Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.
Recombinant lambda phage Charon 4A with repetitive human DNA inserts have been constructed by using cellular DNA from a human-Chinese hamster ovary cell hybrid retaining the complete hamster genome and a single human chromosome 12. One recombinant phage, 12-11, contains several repetitive sequences, each with a different repetition pattern in the human genome. A 2.2-kilobase (kb) EcoRI fragment of this phage was subcloned in pBR325. This sequence has fewer than 5,000 copies in the human genome and does not cross-hybridize with Chinese hamster DNA. When the labeled 2.2-kb probe was hybridized to human chromosome 12 DNA digested with EcoRI, there was an intense band at the 2.2-kb position and a series of other discrete bands. The band pattern at positions other than 2.2 kb appears to be distinct for each human chromosome. The 2.2-kb fragment is composed of at least three subregions. The ends of the fragment are repeated more frequently in the genome than is the middle portion. Hybridization of chromosome 12 DNA with probes made to these subregions yielded simpler band patterns. By using a series of cell hybrids containing various deletions of human chromosome 12, five sequences related to the 2.2-kb fragment have been assigned regionally to a specific portion of the short arm of chromosome 12. These results demonstrate that certain repetitive sequences in the human genome can be used as genetic markers and may permit detailed regional mapping of human chromosomes.
A procedure has been described for micro- dissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromo- somes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repet- itive sequences and 58% contained single or low-copy se- quences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for pro- cesses such as (i) isolating corresponding yeast artificial chro- mosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) construct- ing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technol- ogy for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to
To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.
The gene encoding the beta-amyloid precursor protein has been assigned to human chromosome 21, as has a gene responsible for at least some cases of familial Alzheimer disease. Linkage studies strongly suggest that the beta-amyloid precursor protein and the product corresponding to familial Alzheimer disease are from two genes, or at least that several million base pairs of DNA separate the markers. The precise location of the beta-amyloid precursor protein gene on chromosome 21 has not yet been determined. Here we show, by using a somatic-cell/hybrid-cell mapping panel, in situ hybridization, and transverse-alternating-field electrophoresis, that the beta-amyloid precursor protein gene is located on chromosome 21 very near the 21q21/21q22 border and probably within the region of chromosome 21 that, when trisomic, results in Down syndrome.
