Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.     

Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer.

Binding sites for six distinct nuclear factors on the 75-base-pair repeat of the Moloney murine leukemia virus enhancer have been identified by an electrophoretic mobility shift assay combined with methylation interference. Three of these factors, found in WEHI 231 nuclear extracts, which we have named LVa, LVb, and LVc (for leukemia virus factors a, b, and c) have not been previously identified. Nuclear factors that bind to the conserved simian virus 40 corelike motif, the NF-1 motif, and the glucocorticoid response element were also detected. Testing of multiple cell lines showed that most factors appeared ubiquitous, except that the NF-1 binding factor was found neither in nuclear extracts from MEL cells nor in the embryonal carcinoma cell lines PCC4 and F9, and core-binding factor was relatively depleted from MEL and F9 nuclear extracts.     

Augmentation of c-fos mRNA expression by activators of protein kinase C in fresh, terminally differentiated resting macrophages.

Expression of c-fos mRNA was investigated in fresh, normal peritoneal macrophages (M phi), which are terminally differentiated, nonproliferating cells. The levels of c-fos mRNA were dramatically increased by stimulation with phorbol myristate acetate (PMA), calcium ionophore, or 1-oleoyl-2-acetoyl glycerol (OAG). Induction of c-fos mRNA by all the above agents followed similar kinetics, with a peak of mRNA 30 min after stimulation. These results demonstrate that c-fos mRNA can be augmented in fresh, terminally differentiated cells. Since the stimuli increasing c-fos mRNA are direct or indirect activators of protein kinase C, our data suggest that in M phi c-fos mRNA is controlled by protein kinase C activation. PMA, calcium ionophore, and OAG were biologically active in M phi. PMA and calcium ionophore induced respiratory burst and tumoricidal activity, respectively, whereas OAG and PMA were chemotactic for M phi. Interferons beta and gamma, potent M phi activators eliciting tumoricidal activity, did not alter the levels of c-fos mRNA. These results indicate that c-fos mRNA augmentation is a stimulus-specific rather than a function-specific response connected to activation of protein kinase C.     

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.