Localization of the DER/flb protein in embryos: implications on the faint little ball lethal phenotype.

Antibodies were raised against the Drosophila EGF receptor homolog (DER) and used for immunohistochemical analyses of Drosophila embryos. We found that DER is localized in a wide array of embryonic tissues, displaying a dynamic pattern of expression. DER appears to be expressed in all cells at the cellular blastoderm and gastrula stages. In extended-germ-band embryos, it is found predominantly in the mesoderm and the head. Finally, in retracted-germ-band embryos, DER immunoreactivity is most pronounced at sites of somatic muscle attachments and along the ventral midline of the CNS. We have thus observed that DER is expressed in the diverse tissues which are affected in the DER faint little ball (flb) embryonic lethal phenotype. The different pattern and extent of expression in each tissue suggests that the disparate aspects of the flb phenotype may result from different mechanisms of DER function. To understand the basis for the CNS phenotype of DER/flb mutants, we have closely followed the collapse of the CNS in mutant embryos. Our observations on the evolution of the final CNS phenotype, in combination with the temporo-spatial pattern of appearance of DER in the ventral neuroepithelium, suggest that this receptor participates in the second phase of neuron-glia interactions, namely in stabilization of the ladder-like CNS scaffolding formed by outgrowth of pioneer axonal processes along the glial pre-pattern.

Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain.

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v-erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine-kinase receptor family.

Replication of adenovirus and SV40 chromosomes in vitro.

As an approach to studying the mechanisms involved in the replication of eukaryotic chromosomes, we have developed and characterized cell-free replication systems for the animal viruses, adenovirus and SV40. In this report we summarize recent work on the proteins required for the initiation of DNA synthesis in these two systems. The adenovirus origin of DNA replication was shown to consist of three functionally distinct sequence domains. Cellular proteins that specifically recognize each of these domains were purified and characterized. Initiation of adenovirus DNA replication was reconstituted from two virus-encoded and three cell-encoded factors. The SV40 origin of replication consists of a 65 base pair DNA segment that contains a high affinity binding site for the viral initiation protein T antigen. Evidence is presented that the first step in initiation of SV40 DNA replication involves the specific binding of T antigen to the origin, followed by the local unwinding of the two strands of the template. The unwinding reaction is specific for DNA templates containing the SV40 origin and requires ATP hydrolysis. In addition to T antigen, efficient unwinding requires a cellular factor(s) that can be replaced by the single-stranded DNA binding protein of Escherichia coli. These results indicate that the recently discovered helicase activity of T antigen plays a central role in initiation of viral DNA synthesis.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.

Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors.

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.

Structure and function of the adenovirus origin of replication.

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.

Galactosyl transferase activity in rat bladder transitional cell carcinoma lines and in exfoliated cells in urine during carcinogenesis and reversible hyperplasia.

Cultured cells of rat bladder transitional cell carcinoma line AY-27, in suspension, were assayed for galactosyl transferase (GT) by measurement of the transfer of [3H]galactose from uridine diphosphate-[3H]galactose to desialylated ovine submaxillary mucin (OSM-NANA). The assay was optimized with respect to time and to protein, uridine diphosphate galactose, OSM-NANA and Triton X-100 concentrations. This assay was then applied weekly to suspensions of exfoliated bladder cells collected from urines of rats fed the bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, and of control rats. Increases in activity over controls appeared 42 weeks after feeding the carcinogen, at a stage when bladder tumors were already microinvasive or deeply invasive, and activities at 52 weeks were about 10-fold greater than normal values. In contrast, a bladder cytotoxic agent inducing reversible hyperplasia was injected into rats, and exfoliated cells were collected from urines: these cells showed no greater GT activity than normal. Bladder tumor tissue from a transplanted tumor had the same high specific enzymatic GT activity as exfoliated cells from tumor-bearing rats.

Galactosyl transferase activity in human transitional cell carcinoma lines and in benign and neoplastic human bladder epithelium.

Cultured cells of human transitional cell carcinoma line MGH-U1, in suspension, were assayed for galactosyl transferase by measurement of the transfer of [3H]galactose from uridine diphosphate:[3H]galactose to desialylated ovine submaxillary mucin. The assay was optimized with respect to time and to protein, uridine disphosphate:galactose, desialyated ovine submaxillary mucin, and Triton X-100 concentrations. This assay was then applied to fresh specimens of benign, inflamed, and neoplastic bladder epithelium from 33 patients who under went cold-cup biopsies at cytoscopy. Transitional cell carcinoma specimens gave values in the range of 24.7 to 184.8 cpm [3H]galactose transferred per microgram protein per hr [72.0 +/- 44.7 (S.D.); n = 25]; normal and inflamed specimens ranged from 0.8 to 46.1 cpm/microgram protein per hr [8.3 +/- 8.4 (S.D.); n = 35]. By using a known method of cell rupture, cell ghosts, representing cell-surface membranes, were isolated both from the cultured cell line and from two biopsy specimens of transitional cell carcinoma. Although a complete enzymatic and electron microscopic analysis was not undertaken, the coincidence of an enzyme marker with the cell ghost fraction containing the elevated galactosyl transferase made it appear probable that this enzyme is located in the cell surface.