Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.     

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.     

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Effects of protein perturbants on phospholipid bilayers

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.     

Augmentation of proliferation and in vitro production of cytotoxic cells by 2-ME in the rat.

Low concentrations (10(-5) to 10(-8) M) of 2-mercaptoethanol (2-ME) greatly enhance the proliferation of allogeneic cells in the rat mixed lymphocyte culture (MLC). Studies were undertaken to determine the mode of action of 2-ME. MLC proliferation can occur in the absence of serum proteins (fetal calf serum, FCS) only if 2-ME is present; however, a synergistic effect is present with FCS plus 2-ME, with a 3-fold increase in 3HTdR incorporation with FCS concentrations as low as 0.1%. Kinetic studies show no shift in the peak of proliferation (92 hr) when comparing cultures with and without 2-ME; however, 2-ME-supplemented cultures have significant 3HTdR uptake at 24 hr, and the peak amount of uptake at 92 hr is two to four times higher. Delayed addition of 2-ME until 92 and 166 hr produces a further increase in 3HTdR uptake, indicating that the entire effect is not expressed at the time of allogeneic recognition. L-ascorbic acid, another reducing agent which lacks sulfhydryl groups, elicits a much lower effect on DNA synthesis than does 2-ME. The cytotoxicity of cells harvested from MLC supplemented with 2-ME is increased without loss of target specificity, whereas the same concentration of 2-ME has no direct effect upon the cytotoxicity assay except at higher concentrations where 2-ME suppresses cytotoxicity.     

Composition and spatial heterogeneity of submersed vegetation in a softwater lake in Wisconsin

Big Muskellunge Lake, a softwater lake (pH 7.5–8.0, alkalinity 0.36 meq/L) in northern Wisconsin, harbors a diverse (25 species) submersed macrophyte vegetation. The present submersed flora combines species generally thought distinct in their ecological affinities, and is very similar to that reported in 1935. The only differences are the apparent loss of three previously infrequent Potamogeton species, and the addition to the flora of two infrequent Potamogeton species and the now abundant Elodea canadensis. Dramatic differences in composition and pattern of the vegetation occurred between sites of contrasting exposure and sediment type. Substantial compositional variation, at scales ranging from tens of centimeters to tens of meters, occurred along transects of contiguous quadrats at uniform depth and exposure. Analyses of species sequences along line transects detected significant segregation of species that is most plausibly explained by biotic factors such as colonization, clonal growth, and competition. Heterogeneity at small scales of a few meters or less is common in Big Muskellunge Lake. Such pattern has been neglected in macrophyte ecology, in spite of its significance for neighbor relationships which may critically influence growth and reproduction of aquatic plants.