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.     

Retrovirus-mediated transfer of an adenovirus gene encoding an integral membrane protein is sufficient to down regulate the receptor for epidermal growth factor.

We have used retrovirus-mediated gene transfer to introduce sequences encoding a 10,400-molecular-weight (10.4K) adenovirus protein previously shown to down regulate the receptor for epidermal growth factor (EGF) into two murine cell lines that possess human EGF receptors (EGF-Rs). Assays for receptor expression showed that acute infection resulted in rapid, constitutive down regulation of the EGF-R via a pathway that appears to be endosome mediated. This represents the first demonstration that 10.4K expression in the absence of other virus-encoded proteins is sufficient to elicit this response. The usefulness of this approach for the study of 10.4K-mediated signal transduction in cells with a nontransformed phenotype is discussed.     

Skeletal muscle phenotypes initiated by ectopic MyoD in transgenic mouse heart.

Forced expression of the myogenic regulatory gene MyoD in many types of cultured cells initiates their conversion into skeletal muscle. It is not known, however, if MyoD expression serves to activate all or part of the skeletal muscle program in vivo during animal development, nor is it known how limiting the influences of cellular environment may be on the regulatory effects of MyoD. To begin to address these issues, we have produced transgenic mice which express MyoD in developing heart, where neither MyoD nor its three close relatives--myogenin, Myf-5, and MRF4/herculin/Myf-6--are normally expressed. The resulting gross phenotype in offspring from multiple, independent transgenic founders includes abnormal heart morphology and ultimately leads to death. At the molecular level, affected hearts exhibit activation of skeletal muscle-specific regulatory as well as structural genes. We conclude that MyoD is able to initiate the program that leads to skeletal muscle differentiation during mouse development, even in the presence of the ongoing cardiac differentiation program. Thus, targeted misexpression of this tissue-specific regulator during mammalian embryogenesis can activate, either directly or indirectly, a diverse set of genes normally restricted to a different cell lineage and a different cellular environment.     

c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.     

The formation of ES of cytochrome-c peroxidase: a comparison with lactoperoxidase and horseradish peroxidase

The activation energy for the formation of the first red compound, ES, for cytochrome-c peroxidase (ferrocytochrome-c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) by i-propyl hydroperoxide and the rate constants for the formation of ES with various hydroperoxides have been determined. Multivariate data analysis by the partial least-squares model in latent variables has been used to compare the rate constants with the corresponding rate constants for the formation of compound I from lactoperoxidase and two isoenzymes of horseradish peroxidase. The results show that the rate of formation of ES from cytochrome-c peroxidase is highly correlated with the pKa of the hydroperoxides. The activation energy for the formation of ES with i-propyl hydroperoxide is close to the corresponding value for hydrogen peroxide.     

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.     

Relationships between induction of anesthesia and mitotic spindle disturbances studied by means of principal component analysis

A dataset comprising the activity of 30 compounds in 4 biological tests--anesthesia of tadpoles, anesthesia of frog heart, abnormal growth and spindle disturbances in Allium root tips--was re-evaluated by means of principal component analysis. A two-component model is required to explain the variation in biological activity of the compounds. It is found that abnormal growth is different from the other biological responses. When this test is excluded, as much as 90% of the variation is explained by a one-component model, the determining factor most probably being the lipophilic character of the compounds. Mammalian mitotic cells respond in a similar way to mitotic cells of Allium root tips. It is suggested that possible regularities in the dose-response relationships for anesthesia, teratogenic effects and generation of abnormal chromosome numbers require further exploration.