The human leukemia cell line HL-60 can be induced to differentiate in vitro, permitting a recombinant DNA analysis of the complex series of differentiation steps leading from promyelocytes to either macrophages or granulocytes. We have isolated many genes which are strongly regulated. Characterization during the past year included discovery of a new human tubulin gene and the first human ferritin L subunit clones among the regulated genes as well as a gamma-actin with an amino acid substitution in a sequence normally highly conserved. Mutant beta-actin genes have been implicated as important in affecting oncogenic or metastatic potential. This is the first indication that gamma-actin alterations may have similar effects. Our main emphasis remains the development of a system for using site-directed mutagenesis to study commitment and terminal differentiation in human cells. We have now succeeded in obtaining efficient transfection of HL-60 cells with the pSV2neo vector. By cotransformation, tagged versions of genomic clones for genes which show strong regulation during HL-60 differentiation have also been inserted. Several tagged genes are expressed at high levels and show appropriate regulation. This suggests that we have succeeded in developing the first system permitting the use of site-directed mutagenesis to study the irreversible gene regulation changes occurring during the terminal differentiation of human cells. At the same time we have obtained evidence suggesting that gene regulation during myeloid terminal differentiation involves a complex series of gene switching events. Instead of a single coordinate turn-on, as has been seen in muscle or reticulocyte differentiation, we see a complex series of changes in the levels of different mRNAs. Translational regulation is also important, and in the case of the alpha-tubulin mRNA we have identified an unusual sequence in the 5' untranslated region which may explain a very strong translational regulation. (M)
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