The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

Microscopic mechanism of DNA damage searching by hOGG1

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine (^oG) DNA glycosylase 1 (hOGG1), which repairs mutagenic ^oG lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a covalent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic parameters describing the DNA search of hOGG1 were derived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dynamic multidimensional diffusion paths to scan DNA.     

Changes in the levels of wheat- and barley-germ agglutinin during embryogenesis in vivo,in vitro and during germination

Radioimmunoassay has been used to measure levels of wheat-germ agglutinin and barley-germ agglutinin during embryogenesis and germination. The two lectins exhibited similar patterns of accumulation during grain maturation in vivo and both decreased to low levels after imbibition of harvest-ripe grains for 3 d. Precocious germination of immature wheat and barley embryos excised and cultured in vitro could be prevented either by inclusion of abscisic acid or mannitol in the culture medium. Changes in the level of wheat-germ agglutinin induced by in vitro culture depended on the maturation stage of the embryo. No direct correlation was found between application of exogenous abscisic acid and accumulation of the lectin.     

Detection and characterization of low abundance cellular proteins that specifically increase upon loss of the metastatic phenotype

Human epidermoid carcinoma (HEp-3) cells are highly tumorigenic and metastatic in vivo, but their metastatic phenotype is progressively and uniquely lost upon serial passage in vitro. The nonmetastatic phenotype is fully reversible to the highly metastatic state when HEp-3 cells are passaged back in vivo. To study the complex process of metastasis and its possible negative regulation by specific gene products, the expression of specific proteins between the highly metastatic and nonmetastatic HEp-3 cells was investigated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and subsequent computer assisted analyses. Concomitant with the in vitro loss of metastatic potential of HEp-3 cells is the elevated expression of a subset of low abundance proteins detectable in 2D-PAGE but not apparent in high resolution one dimensional PAGE. When the HEp-3 cells revert to the metastatic state, the expression of these proteins declines. The increased abundance of four distinct proteins directly correlates with the loss of the metastatic phenotype: two of the four proteins are associated with isolated cellular membranes (36kD, pl 5.7; 22kDa, pl 5.6), on protein fractionates with the cytoplasm (65kD, pl 6.2), and one protein is enriched in the nuclei fraction (32kD, pl 5.8). These data indicate that computer-assisted analysis of highly sensitive, large-format, 2D-PAGE can be used to identify specific proteins in subcellular compartments that are candidates for negative regulators of the metastatic process. © 1993 Wiley-Liss, Inc.