Endogenous expression of E1A in human cells enhances the effect of adenovirus E3 on class I major histocompatibility complex antigen expression.

Group C human adenovirus (Ad) serotypes (e.g., Ad type 2 [Ad2] and Ad5) cause persistent infections in humans. One explanation for Ad persistence is an ineffective cytotoxic T-lymphocyte response due to diminished cell surface expression of class I major histocompatibility antigen (MHC Ag) on Ad-infected cells, an effect mediated by the Ad E3 19-kDa glycoprotein (E3 effect). However, we previously reported that, except for the Ad5 E1-transformed human cell line 293, a variety of human lymphoid, epithelial, and fibroblastic cells are resistant to the E3 effect during Ad5 infection (J. M. Routes and J. L. Cook, J. Immunol. 144:2763-2770, 1990). The present study tested the hypothesis that endogenous expression of E1A proteins in 293 cells sensitizes cells to this E3 effect, resulting in an enhanced downregulation of surface class I MHC Ag expression following Ad5 infection. Human epithelial and fibroblastic cells expressing E1A gene products for at least 72 h exhibited an enhanced E3 effect following Ad5 infection that was independent of baseline levels of surface class I MHC Ag expression and of E1A induction of E3 19-kDa glycoprotein expression. There was a direct correlation between the level of endogenous E1A expressed and the magnitude of the E3 effect. We postulate that the in vivo existence of cells stably expressing either E1A proteins or E1A-like activities in the microenvironment of Ad5 infection provides a reservoir of Ad-infected cells that is relatively protected from the virus-specific cytotoxic T-lymphocyte response, thereby favoring Ad persistence in humans.

Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis-elements and trans-acting factors.

The primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania rostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal, trans-acting factor present in nuclear extracts of nodules. These results suggest a conserved mechanism for nodule specific induction of lb genes in leguminous plants.

Developmentally regulated transcription of Dictyostelium discoideum plasmid Ddp1.

The presence of high copy number plasmid DNA has been described for several independent wild isolates of the lower eukaryote Dictyostelium discoideum, a model organism in developmental biology. The function of these plasmids has remained unclear so far. To understand the possible functions of the plasmids we have analyzed the transcription of one plasmid, Ddp1, in the wild strains NC4 and V12. The plasmid is transcribed in an identical fashion in both strains. Ddp1 codes for three RNA species in growth-phase cells, at least two of which are present in the poly(A) fraction. RNA isolated from cells of different developmental stages revealed the existence of five more transcripts, the majority of them being found in the poly(A) fraction. These transcripts are present at distinct time points during development. Both growth-phase and development-specific transcripts were found exclusively in Ddp1-containing strains. Mapping of the transcripts to the plasmid showed that some of them originated from the same area. These results indicated an overlapping of their coding regions.

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.

Presence of nuclear associated plasmids in the lower eukaryote Dictyostelium discoideum.

High copy number plasmids have been identified in six out of 25 wild-type strains of the cellular slime mould Dictyostelium discoideum, a model organism in developmental biology (Loomis, 1982). The characterization of three plasmids, from the NC4 (Ddp1), WS380B (Ddp2) and OHIO (Ddp3) wild isolates, is presented here. We show that they are nuclear associated and non-homologous to the mitochondrial DNA and extrachromosomal ribosomal DNA.

Identification of an endogenous plasmid in Dictyostelium discoideum.

A plasmid has been discovered in a strain of the eukaryote, Dictyostelium discoideum, which has an unstable, non-chromosomal, cobalt resistance phenotype. The plasmid, termed Ddp1, is 13.5 kbp in size and is found in the nucleus. It has an A-T content typical of Dictyostelium DNA as judged by its restriction enzyme digestion pattern, and it is not related to either mitochondrial or ribosomal DNA. Similar or identical plasmids have been found in two original, cobalt-sensitive, isolates, NC4 and V12, but no plasmid was detected in three other isolates (WS472, WS526, WS584). The plasmid codes for non-essential functions since it is absent from the latter isolates, and it is lost from mutant strains which are capable of axenic growth.

Chromosome rearrangements in Dictyostelium discoideum.

A tandem duplication (D350(III,III] of the whiB to radB interval of linkage group III has been characterized. The gene order on the duplication-bearing chromosome is: centromere, whiB500, radB+, whiB+, radB24, bsgA5, acrC4. Slow-growing, duplication-bearing strains (yellow-spored, radiation-resistant) produced four classes of faster growing sectors involving the whiB and radB loci: white-spored, radiation-sensitive (whiB500, radB24); white-spored, radiation-resistant (whiB500, radB+); yellow-spored, radiation-sensitive (whiB+, radB24); and yellow-spored, radiation-resistant. The first three classes can be explained as the products of single recombination events in which one copy of the whiB to radB interval was lost. The yellow-spored, radiation-resistant sectors probably arose by mutation elsewhere in the genome, but alternatively may represent multiple recombination events or deletion of part of one copy of the duplicated region. Loss of the duplicated segment was enhanced by irradiation with ultraviolet light (254 nm). Heterozygosity for a DNA repair mutation at the radB locus may have been involved in the formation of the duplication. It is proposed that translocations are a major cause of nonrandom segregation patterns such as the cosegregation of unlinked markers in Dictyostelium discoideum. Translocations involving all known linkage groups are tabulated and DNA damage by N-methyl-N'-nitro-N-nitrosoguanidine is implicated in the formation of translocations in D. discoideum.