Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.     

Protein threading by recursive dynamic programming.

We present the recursive dynamic programming (RDP) method for the threading approach to three-dimensional protein structure prediction. RDP is based on the divide-and-conquer paradigm and maps the protein sequence whose backbone structure is to be found (the protein target) onto the known backbone structure of a model protein (the protein template) in a stepwise fashion, a technique that is similar to computing local alignments but utilising different cost functions. We begin by mapping parts of the target onto the template that show statistically significant similarity with the template sequence. After mapping, the template structure is modified in order to account for the mapped target residues. Then significant similarities between the yet unmapped parts of the target and the modified template are searched, and the resulting segments of the target are mapped onto the template. This recursive process of identifying segments in the target to be mapped onto the template and modifying the template is continued until no significant similarities between the remaining parts of target and template are found. Those parts which are left unmapped by the procedure are interpreted as gaps.The RDP method is robust in the sense that different local alignment methods can be used, several alternatives of mapping parts of the target onto the template can be handled and compared in the process, and the cost functions can be dynamically adapted to biological needs.Our computer experiments show that the RDP procedure is efficient and effective. We can thread a typical protein sequence against a database of 887 template domains in about 12 hours even on a low-cost workstation (SUN Ultra 5). In statistical evaluations on databases of known protein structures, RDP significantly outperforms competing methods. RDP has been especially valuable in providing accurate alignments for modeling active sites of proteins.RDP is part of the ToPLign system (GMD Toolbox for protein alignment) and can be accessed via the WWW independently or in concert with other ToPLign tools at http://cartan.gmd.de/ToPLign.html.     

Bacteriophage-resistance mechanisms examined by transfection of Lactococcus lactis subsp. lactis 4513-5 mediated by high voltage electroporation

Summary Phage adsorption tests and transfection by electroporation were carried out to decide whether phage-resistance in Lactococcus lactis subsp. lactis strain 4513-5 is based on intracellular or extracellular mechanisms. Using high voltage (12.5 kV/cm) electroporation, untreated phage DNA was introduced into phage-sensitive and phage-resistant cells. Since phages showed low adsorption frequencies on resistant bacteria, resistance is localized in the cell wall preventing phage DNA from entering the cell. This is the only mechanism responsible for the resistance of L. lactis subsp. lactis 4513-5 against its homologous phage P4513-K12 and non-homologous phages P05M-13 and P05M-47, but not against phage P530-7 and phage P530-12. In the case of the latter two phage strains, intracellular resistance mechanisms are involved and discussed.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Linking of the human immunoglobulin VK and JKCK regions by chromosomal walking.

The linking of the human VK and JKCK gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between JKCK and the telomer show that none of the major VK gene clusters is located downstream of CK. The distance between the VK and JK genes was found to be 23 kb. The JK proximal VK gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to JKCK and can therefore rearrange only by an inversion mechanism. This finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step.     

Localization, analysis and evolution of transposed human immunoglobulin V kappa genes

The localization of V kappa gene regions to chromosome 2, on which the kappa locus is located, and to other chromosomes is described. The V kappa genes that have been transposed to other chromosomes are called orphons. The finding of two new V kappa genes on chromosome 22 is reported. A V kappa II gene of this region and two V kappa I genes of the Chr1 and the cos 118 regions were sequenced. The two V kappa I orphon sequences and two others that had been determined previously were 97.5% identical, indicating that they may have evolved from a common ancestor by amplification. A model of the evolution of the human V kappa orphons is discussed.     

Location of Oospores in Buckwheat Seed and Probable Roles of Oospores and Conidia of Peronospora ducometi in the Disease Cycle on Buchwheat

Oospores of Peronospora ducometi, the causal agent of downy mildew of buckwheat (Fagopyrum esculentum), were found in the calyx remnant attached to the seed, on the inside of the seedcoat and in the spermoderm layer between the seedcoat and the endosperm. This constitutes a first report documenting the location of oospores in buckwheat seed. Systemic infection of seedlings occurred from oospore-infested seed. Conidial germination was greater at 14°C than 25°C. Systemic infection also occurred as the result of conidial infection of leaves. It is proposed that primary infection of buckwheat occurs by the germination of seed-borne oospores resulting in systemic invasion of the seedling by the germtubes, and followed by conidial formation on the cotyledons. Secondary infection occurs initially from conidia produced on the cotyledons as a result of the systemic infection from seed and subsequently as the result of repeated infections by conidia produced on leaf lesions as the disease progresses up the plant.     

Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase

Resonance Raman spectroscopy has been employed to investigate the molecular bases for the markedly different properties of horseradish oxyperoxidase and oxymyoglobin. The porphyrin core of oxyperoxidase is slightly more expanded with the iron atom closer to the porphyrin plane, and there is greater iron d pi-to-oxygen pi backbonding compared to oxymyoglobin. The iron-oxygen (stretching or bending) bands are observed at 570 and 562 cm-1, respectively, for oxymyoglobin and oxyperoxidase, and the iron-His stretching bands have been tentatively identified at 276 and 289 cm-1, respectively. It is suggested that the stronger iron-His bond in oxyperoxidase facilitates greater iron d pi-to-oxygen pi backdonation by raising the energy of the iron d pi orbitals closer to the energy of the oxygen pi orbitals. This weakens the O-O bond and activates dioxygen for use as an electron acceptor in the peroxidase-oxidase reaction.     

Quantitative study of cell interaction with collagen and fibronectin

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).