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The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time. Sendai virus is used to promote cell hybridization. beta-propriolactone is employed as the inactivating agent of Sendai virus since it produces complete loss of viral infectivity while preserving viral fusion capacity. Cells are fused in monolayer, instead of in suspension, since fixing cells in two dimensions permits one to control cell contacts during the fusion event through the expedient of varying multiplicities of the parental cells and the total cell density. Under the conditions described, a several hundred fold increase in the number of hybrid clones obtained is seen as compared to the controls.

The intrachromosomal localization of three X-linked gene loci (PGK, HGPRT and G6PD) has been determined using a somatic cell genetic approach. A human cell line possessing an X/14 translocation was used as one parent in the formation of human/mouse hybrids. The translocation separates the human X into two parts: Xp and t(Xq14q). The data indicate that all three X-linked loci segregate with the t(Xq14q) rearrangement product thus permitting their assignment to the X chromosome's long arm. Secondary rearrangements and data from other laboratories suggest that the order of the the three markers from the centromere to the distal end of the X long arm is PGK, HGPRT, G6PD. It was also observed that NP, an autosomal locus, segregated with the t(Xq14q) chromosome. This provides strong support for the assignment of NP to 14.

Hybrids between two parental lines of somatic cells have been observed in various genomic multiplicities by ourselves and others. Possible explanations for the higher multiplicities include the fusion of cells in a 1:1 ratio with one genomic set undergoing an asynchronous replication either before or after fusion or the fusion of two or more cells. We now provide evidence for multiple genomic hybrids arising from the fusion of more than two cells in a mixture of three different cell lines. This proof is based on unique chromosomal and biochemical markers characteristic of the three parental cell lines. The distinctive phenotypes of all three lines are expressed in a clonally derived hybrid. Thus, we conclude that long-term, proliferating somatic cell hybrids can arise from the fusion of three or more cells.

Activity of the enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase increases intermittently in synchronized cultures of Chinese hamster cells. During G(1) phase (3 hours after mitosis), midway through S phase (7 hours after mitosis) and again in late S phase (10 hours after mitosis), rapid increases in activity were observed and correlated with a net increase in cell size and total protein. The evidence suggests that this is a general phenomenon which affects a whole population of proteins and may be the expression of an endogenous cellular rhythm.

Independently derived man-mouse somatic cell hybrids and their derivative subclones show a positive correlation between the expression of human lactate dehydrogenase A subunits and the occurrence of the human C-11 chromosome. Data are also presented that confirm the previously reported linkage of the thymidine kinase locus to the E-17 chromosome. A translocation of the E-17 chromosome provides presumptive evidence for the assignment of the thymidine kinase locus to the long arm segment of the E-17 chromosome. This translocation also provides evidence for translocation between man and mouse chromosomes in somatic cell hybrids. A presumptive association between the human phenotype for isocitrate dehydrogenase and the human F group is also described. Identification of specific human chromosomes was achieved by the application of several new cytological techniques: measurement of chromosome arm length, in situ annealing with mouse satellite complementary RNA, constitutive heterochromatin staining with Giemsa, and quinacrine mustard fluorochromatic staining.