Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances.
Transcription factors that are bound specifically to DNA often interact with each other over thousands of base pairs  . Large DNA loops resulting from such interactions have been observed in Escherichia coli with the transcription factors deoR  and NtrC , but such interactions are not, as yet, well understood. We propose that unique protein complexes, that are not present in solution, may form specifically on DNA. Their uniqueness would make it possible for them to interact tightly and specifically with each other. We used the repressor and operators of coliphage lambda to construct a model system in which to test our proposition. lambda repressor is a dimer at physiological concentrations, but forms tetramers and octamers at a hundredfold higher concentration. We predict that two lambda repressor dimers form a tetramer in vitro when bound to two lambda operators spaced 24 bp apart and that two such tetramers interact to form an octamer. We examined, in vitro, relaxed circular plasmid DNA in which such operator pairs were separated by 2,850 bp and 2,470 bp. Of these molecules, 29% formed loops as seen by electron microscopy (EM). The loop increased the tightness of binding of lambda repressor to lambda operator. Consequently, repression of the lambda PR promoter in vivo was increased fourfold by the presence of a second pair of lambda operators, separated by a distance of 3,600 bp. (+info)
A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation.
Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation. (+info)
Novel endotheliotropic herpesviruses fatal for Asian and African elephants.
A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease. (+info)
The haplotype distribution of two genes of citrus tristeza virus is altered after host change or aphid transmission.
Genetic variability of citrus tristeza virus (CTV) was studied using the haplotypes detected by single-strand conformation polymorphism (SSCP) analysis of genes p18 and p20 in six virus populations of two origins. The Spanish group included a CTV isolate and subisolates obtained by graft-transmission to different host species. The other included two subisolates aphid-transmitted from a single Japanese isolate. The homozygosity observed for gene p20 was always significantly higher than that expected under neutral evolution, whereas only three populations showed high homozygosity for p18, suggesting stronger host constraints for p20 than for p18. Sequential transmissions of a Spanish isolate to new host species increased the difference between its population and that of the successive subisolates for gene p18, as estimated by the F statistic. Analysis of molecular variance indicated that variation between both groups of populations was not statistically significant, whereas variations between populations of the same group or within populations were significant for both genes studied. Our data indicate that selection affects the haplotype distribution and that adaptation to a new host can be as important or more as the geographical origin. Variation of the CTV populations after host change or aphid transmission may explain in part the wide biological variability observed among CTV isolates. (+info)
Bacteriophage SPO1 development: defects in a gene 31 mutant.
SPO1 temperature-sensitive mutant ts14-1, located in cistron 31, has a DD (DNA synthesis-delayed) phenotype at 37 degrees C and produces progeny in a stretched program. At 44 degrees C it behaves as a DO (DNA synthesis-defective) mutant and shuts off the viral RNA synthesis about 10 min after infection. The thermal sensitivity of this mutant is due to the inactivity of gp-31 (the product of gene 31) at 44 degrees C. However, gp-31 is synthesized at that temperature and partly recovers its activity at 37 degrees C. Only 5 min at the permissive temperature is enough to trigger the continuation of the phage program and to produce progeny. The partial defect at 37 degrees C and the expansion of the middle program together with the pleiotropic defects at the nonpermissive temperature could be suitable for the study of the controls involved in bacteriophage development. (+info)
Evidence that the neck appendages are adsorption organelles in Bacillus subtilis bacteriophage phi29.
A mutant of Bacillus subtilis unable to adsorb phage phi29 efficiently has been isolated. This mutant can be infected by host range mutants of the phage. Since the host range mutations map in cistron 12, which codes for neck appendage protein, this would tend to confirm that these organelles are involved in viral adsorption. (+info)
Control of corynebacteriophage reproduction by heteroimmune repression.
Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells. (+info)
Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA.
The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map. (+info)