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.     

Interannual variability of phytoplankton productivity and related parameters in Lake Constance: no response to decreased phosphorus loading?

In meso-eutrophic Lake Constance (Germany-Austria-Switzerland),phytoplankton bioraass, pigments and water transparency, aswell as primary productivity, have been followed between 1980and 1989. During this period, municipal phosphorus loading declinedsignificantly. Since 1981, soluble reactive phosphorus (SRP)concentrations during deep lake mixing have decreased from 3.0to currently 1 6 mmol m^–3 at a rate of 7% year^–1.Nitrate concentrations, by contrast, continued to rise. Duringthe period of maximum phosphorus loading, flushing through theoutlet and sedimentation were about equally important sinksof phosphorus from the euphotic zone. Recently, however, sedimentationand subsequent burial of P in the bottom deposits contributedabout three-quarters to the overall P-losses from the systemMain reasons for this shift are unchanged settling fluxes ofphosphorus out of the euphotic zone and decreasing concentrationsof total phosphorus in the water. Only during spring, do concentrations of soluble reactive phosphoruswithin the euphotic zone decrease in proportion to the formationof particulate organic matter. Later during the season, euphoticSRP concentrations continue to be low but are no longer matchedby high plankton biomass because phosphorus is efficiently removedby settling of particles In spite of the observed dramatic decreasein phosphorus loading since 1980, chlorophyll concentrationsand water transparency, as well as annual phytoplankton productivity(300 g C m^–2), have not shown a consistent downward trend.However, the intensity of phosphorus regeneration within theeuphoric zone, which can be used as a measure of the degreeof nutrient limitation, is likely to have increased significantlyThe most probable explanation for the insensitivity of importanttrophic state indicators to reduced nutrient loading is that,in Lake Constance, biomass accumulation to a greater extentis controlled by losses, mainly grazing by zooplankton and sedimentation,than by primary resources. This is concluded from the observationthat phytoplankton biomass always falls far short of the nutrient-dependentcarrying capacity of the system.     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

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.     

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.