Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+.

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.     

Floral scent variation in the Pyrola rotundifolia complex in Scandinavia and Western Greenland

The variation in floral scent composition within and among populations of four taxa belonging to the Pyrola rotundifolia complex was analyzed using Principal Component Analysis. Two major groups were recognized, P. norvegica and P. grandiflora on the one side and P. rotundifolia s. str. on the other. Benzaldehyde dominated the scent of the first group and methoxy benzenes and phenyl propanoids that of the second group. A large variation in the floral scent chemistry was found both within some of the studied populations as well as among them. The floral scent composition of P. rotundifolia ssp. maritima was no more different from P. rotundifolia ssp. rotundifolia , than the differences between populations of P. rotundifolia ssp. rotundifolia . The findings are in partial agreement with the current delimitation of the taxa in the P. rotundifolia complex.     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Null alleles at two lactate dehydrogenase loci in rainbow trout are associated with decreased developmental stability

Previous studies with rainbow trout (Oncorhynchus mykiss) have shown that allozymic heterozygotes have increased developmental stability, as measured by reduced fluctuating bilateral asymmetry. In this paper, we examine the phenotypic effects of null alleles at two lactate dehydrogenase (LDH) loci. If the association between allozymic heterozygosity and developmental stability is due largely to linked chromosomal segments, then we would expect null allele heterozygotes to have increased developmental stability. In contrast, heterozygotes for LDH null alleles in three populations have reduced developmental stability. This suggests that the reduction in enzyme activity at these loci is having a deleterious effect on development that is strong enough to mask any beneficial effects that may be associated with heterozygosity for these chromosomal segments. The LDH loci examined in this study are members of two different paralogous pairs of duplicate genes produced by the polyploidization of the ancestral salmonid genome. The apparent deleterious effects of these null alleles in heterozygotes could retard the possible loss of duplicate gene expression.     

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.