Using either highly purified RNA polymerase II (pol II) elongation complexes assembled on oligo(dC)-tailed templates or promoter-initiated (extract-generated) pol II elongation complexes, the precise 3" ends of transcripts produced during transcription in vitro at several human c- and N- myc pause, arrest and termination sites were determined. Despite a low overall similarity between the entire c- and N- myc first exon sequences, many positions of pol II pausing, arrest or termination occurred within short regions of related sequence shared between the c- and N- myc templates. The c- and N- myc genes showed three general classes of sequence conservation near intrinsic pause, arrest or termination sites: (i) sites where arrest or termination occurred after the synthesis of runs of uridines (Us) preceding the transcript 3" end, (ii) sites downstream of potential RNA hairpins and (iii) sites after nucleotide addition following either a U or a C or following a combination of several pyrimidines near the transcript 3" end. The finding that regions of similarity occur near the sites of pol II pausing, arrest or termination suggests that the mechanism of c- and N- myc regulation at the level of transcript elongation may be similar and not divergent as previously proposed.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a membrane-associated GTPase with significant similarity to the family of ARFs. We have recently shown that ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor Sec7-1/cytohesin and inhibits the ARF/Sec7-dependent activation of phospholipase D in a GTP-dependent manner. In order to further analyze the function of ARFRP1, we cloned the mouse Arfrp1 gene and generated Arfrp1 null-mutant mice by gene targeting in embryonic stem cells. Heterozygous Arfrp1 mutants developed normally, whereas homozygosity for the mutant allele led to embryonic lethality. Cultured homozygous Arfrp1 null-mutant blastocysts were indistinguishable from wild-type blastocysts. In vivo, they implanted and formed egg cylinder stage embryos that appeared normal until day 5. Between embryonic days 6 and 7, however, apoptotic cell death of epiblast cells occurred in the embryonic ectoderm during gastrulation, as was shown by histological analysis combined with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Epiblast cells that would normally differentiate to mesodermal cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. In contrast, the development of extraembryonic structures appeared unaffected. Our results demonstrate that ARFRP1 is necessary for early embryonic development during gastrulation.ADP-ribosylation factors (ARFs) are GTP-binding proteins that are involved in multiple steps of membrane trafficking and regulation of phospholipase D (PLD) (5,15,20,24,29). ARFRP1 (ARF-related protein 1), previously designated ARP (25), is a membrane-associated 25-kDa GTPase with remote similarity to ARF and ARF-like protein (33 and 39% identical amino acids to ARF1 and ARF-like 3, respectively). ARFRP1 contains all characteristic sequence motifs involved in nucleotide binding and GTP hydrolysis. Compared with other GTPases, guanine nucleotide exchange of recombinant ARFRP1 is slow but GTPase activity is high in the absence of an activating protein. In contrast to ARF and ARF-like proteins, ARFRP1 lacks the N-terminal myristoylation motif (glycine 2) necessary for membrane association. However, ARFRP1 is predominantly located in the plasma membrane and is absent from the cytosol (25), whereas ARF proteins shuttle between membranes and the cytosol, depending on the bound nucleotide (3). Previous studies have suggested that ARFRP1 is involved in a pathway inhibiting the ARF-controlled activity of PLD (26). ARFRP1 binds the ARF-specific nucleotide exchange factor Sec7-1/cytohesin in a GTP-dependent manner and inhibits the ARF/Sec7-dependent activation of PLD. In addition, transfection of HEK-293 cells with a constitutively active mutant of ARFRP1 inhibited the PLD stimulation induced by muscarinic acetylcholine receptor-3 and the translocation of ARF from the cytosol to membranes.ARF and ARF-like proteins are highly conserved throughout the evolution of eukaryotes (29). In yeast, five me...

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