Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.     

Chromosomal Organization of Rrna Operons in Bacillus Subtilis

Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion.     

Instability of rRNA operons in Bacillus subtilis.

Many laboratory strains of Bacillus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnJ-rrnW or rrnI-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-aroI region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Ultrastructure of the Flame Bulbs of Urastoma cyprinae (Platyhelminthes, ‘Prolecithophora’, Urastomidae)

The ultrastructure of the flame bulbs of the turbellarian Urastoma cyprinae from Mytilus galloprovincialis in the Mediterranean is described. The nucleus of the terminal cell is located some distance basal to the rootlets of the cilia forming the flame; the cytoplasm contains numerous tubules approximately 54–66 nm in diameter, and vesicles. Thick walled, densely packed rod-like structures coil around each other with a tendency towards longitudinal orientation close to the flame. The rod-like structures tightly surround the basal part of the flame and the distal cytoplasmic tube in the apical part of the flame. Some of them, including the inner predominantly longitudinally directed ones, are continuous with the cytoplasm of the terminal cell, others are continuous with the cytoplasm of the distal cytoplasmic tube. Internal leptotriches arise from the cytoplasm of the terminal cell and intrude between the basal parts of the cilia of the flame. The distal cytoplasmic tube possesses a septate junction. The flame bulb of Urastoma differs distinctly from those known from other Platyhelminthes; implications for the phylogeny of Platyhelminthes are discussed.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

A cladistic analysis of the genera in the subfamily Pudicinae (Nematoda, Trichostrongyloidea, Heligmonellidae).

A parsimony analysis was performed on 37 specific taxa belonging to the subfamily Pudicinae (family Heligmonellidae), which contains parasites mainly from South American caviomorph rodents. Thirteen characters were used from the synlophe (rotation of axis, presence of carene, carene asymmetry, presence of comaretes, single ventral comarete length, ridge discontinuity, ventral ridge numbers, presence of a peculiar posterior synlophe, presence of supernumerary spines) and the male caudal bursa (relative length of rays 9 and 10, caudal bursa type, division of the dorsal ray, divergence of the 10th rays). The cladogram shows a consistency index of 1.0. The subfamily Pudicinae has two synapomorphies. Two suprageneric groups are recognized. Suprageneric group 1 shows one synapomorphy and contains Heligmostrongylus, Fuellebornema, Sciurodendrium and Pseudoheligmosomum; suprageneric group 2 shows two synapomorphies and contains Pudica, Acanthostrongylus, Justinema and Durettestrongylus. Five genera are defined on the basis of synapomorphies. The genera Heligmostrongylus, Sciurodendrium and Pudica which are considered paraphyletic, however, are retained due to lack of knowledge as to their relationships.