Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5 ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

An Experimental Test for Synergistic Epistasis and Its Application in Chlamydomonas

Theoretically, one of the most general benefits of sex is given by its function in facilitating selection against deleterious mutations. This advantage of sex may be deterministic if deleterious mutations affect the fitness of an individual in a synergistic way, i.e., if mutations increase each others' negative fitness effect. We present a new test for synergistic epistasis that considers the skewness of the log fitness distribution of offspring from a cross. We applied this test to data of the unicellular alga Chlamydomonas moewussii. For this purpose, two crosses were made: one between two strains that are presumed to have accumulated slightly deleterious mutations, the other between two strains without a history of mutation accumulation. Fitness was measured by estimating the two parameters of logistic growth in batch culture, the maximum growth rate (r) and the carrying capacity (K). The finding of a negatively skewed distribution for K in the accumulation cross suggests synergism between mutations affecting the carrying capacity, while the absence of skewness for r in both crosses is consistent with independent effects of mutations affecting this parameter. The results suggest a possible alternative explanation for the general observation that sex is related to constant environments, where selection on K predominates, while asexual reproduction is found in more variable environments, where selection on r is more important.     

Physical and genetic characterization of the IncI plasmid R144-drd3

A physical and genetic map of the IncI plasmid R144-drd3 was obtained by determining restriction endonuclease sites and by physical and genetic analysis of cloned fragments, of Tn1 insertion mutants and of deletion mutants.     

Exclusion in IncI-type Escherichia coli conjugations: the stage of conjugation at which exclusion operates

The stage at which exclusion operates in matings between donors belonging to the I-type incompatibility group (IncI) was investigated. Mating between Escherichia coli cells harbouring the I-type plasmid R144 and E. coli cells harbouring the R144-derived recombinant plasmid pRAH308, which causes a hundredfold exclusion, was performed on a membrane filter to test whether mating aggregate formation was disturbed. Besides, level and kinetics of the formation of mating aggregates in mixtures of R144⁺ donor cells and recipient cells carrying plasmid pRAH308 (exclusion-proficient) was compared with the aggregate formation in mixtures of the donor cells and exclusion-deficient recipient cells. Results from these experiments revealed that the exclusion by pRAH308 does not operate at the level of aggregate formation, but acts at the stage of DNA transfer. The exclusion phenomenon by the recombinant plasmid pRAH308 appeared to be representative for exclusion caused by plasmid R144, since essentially identical results were obtained if plasmid R144 was used as exclusion-determining factor.     

Isolation and characterization of Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor.

Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor (ConI-) were isolated and characterized. These mutants are specific in that they are conjugation proficient with other types of donor strains. They have an altered susceptibility to phages and detergents. Chemical analysis of the cell envelopes of mutant strains has shown that the lipopolysaccharide (LPS) is altered and that one major outer-membrane protein is absent. Conjugation experiments in which LPS from wild-type cells was added to a mating mixture, made up with wild-type donor and recipient cells, showed inhibition in transconjugant formation when an I-type donor, but not an F-type donor, was used. This strongly suggests that LPS of the recipient cell is directly involved in the ability to mate with an I-type donor but not with an F-type donor. The mutations are located in the 78- to 82-min region of the E. coli map, with one exception where the mutation maps near or in the galactose operon.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Sorting of sphingolipids in the endocytic pathway of HT29 cells

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed. After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensin-induced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution. By contrast, C6-NBD-sphingomyelin does not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments. Also, in monensin-treated cells no change in the distribution of fluorescence was observed. Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids. In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin.