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Chapter 5,825

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

Deisseroth, A.; Nienhuis, A.; Turner, P.; Velez, R.; Anderson, W.F.; Ruddle, F.; Lawrence, J.; Creagan, R.; Kucherlapati, R.

Cell 12(1): 205-218

1977

ISSN/ISBN: 0092-8674
PMID: 561664
DOI: 10.1016/0092-8674(77)90198-2
Accession: 005824763
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Sixteen human .times. mouse somatic cell hybrids containing a variable number of human chromosomes were used to demonstrate that the human .alpha.-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human .alpha.-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which were characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the .alpha.-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human .alpha.-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human .alpha.-globin sequences, 1 contained chromosome 2 (JFA 14a 5), 3 contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and 2 contained chromosome 5 (AHA 16E and JFA14a 13 5) in > 10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which were previously suggested to contain human globin genes. Only chromosome 16 was present in each one of the 3 hybrid cell clones found to be strongly positive for the human .alpha.-globin gene. Two clones (WAIVA and WAV) positive for the human .alpha.-globin gene and chromosomes 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked human chromosome 16 and the .alpha.-globin gene. These studies establish the localization of the human .alpha.-globin gene to chromosome 16 and represent the 1st assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.

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Related References

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