Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles. Structural studies by gel chromatography,electron microscopy and nuclear magnetic resonance

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn²⁺ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10–20 mM.Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine.Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical.Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215–240 Å, and an almost constant internal volume.Between 30 and 60% of succinylphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.     

Calmodulin is essential for assembling links necessary for exocytotic membrane fusion in Paramecium.

Calmodulin has long been suspected to be involved in calcium-regulated exocytosis but its precise site(s) of action has not yet been identified. In Paramecium, a genetic approach to the problem is possible as in vivo-selected mutations in the calmodulin gene that prevent the activation of some channels have been characterized. Three of these calmodulin mutants were examined for exocytotic capacity and the mutant cam1 was found to be defective for exocytosis at 35 degrees C. The loss of exocytotic capacity in cam1 cells can be restored by transformation with the wild-type calmodulin gene, demonstrating that its exocytotic lesion is indeed due to the mutation in the calmodulin gene. The cam1 mutant displays abnormal exocytotic sites at the non-permissive temperature: it lacks the links ('rosettes' of intramembranous particles in the plasma membrane and the fibrous 'connecting material') which normally connect plasma and trichocyst membranes. Upon shift of cam1 cells from the permissive to a non-permissive temperature, performed sites remain functional. These results demonstrate that calmodulin is necessary for the assembly of these links at the exocytotic site. These results do not, however, exclude the possibility of calmodulin also being involved in Ca(2+)-dependent steps of the stimulus-exocytosis coupling.     

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics

This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.      

DNA fingerprints from hypervariable mitochondrial genotypes

Conventional surveys of restriction-fragment polymorphisms in mitochondrial DNA of menhaden fish (Brevoortia tyrannus/patronus complex) and chuckwalla lizards (Sauromalus obesus) revealed exceptionally high levels of genetic variation, attributable to differences in mtDNA size as well as in restriction sites. The observed probabilities that any two randomly drawn individuals differed detectably in mtDNA genotype were 0.998 and 0.983 in the two species, respectively. Thus, the variable gel profiles provided unique mtDNA "fingerprints" for most conspecific animals assayed. mtDNA fingerprints differ from nuclear DNA fingerprints in several empirical respects and should find special application in the genetic assessment of maternity.      

The apportionment of dinucleotide repeat diversity in Native Americans and Europeans: a new approach to measuring gene identity reveals asymmetric patterns of divergence

The purpose of this paper is to assess the extent of gene identity and differentiation at 33 dinucleotide repeat loci (377 total alleles) within and among three European and three Native American populations. In order to do this, we show that a maximum-likelihood method proposed for phylogenetic trees (Cavalli-Sforza and Piazza 1975) can be used to estimate gene identity (Nei 1987) with respect to any hierarchical structure. This method allows gene differentiation to be evaluated with respect to any internal node of a hierarchy. It also allows a generalization of F- and G-statistics to situations with unequal expected levels of differentiation. Our principal finding is that levels of genetic differentiation are unique to specific populations and levels of nesting. The populations of European origin show very little internal differentiation; moreover, their continental average is close to the total population defined by the aggregate of Europeans and Native Americans. By contrast, the Native American populations show moderate levels of internal differentiation, and a great distance between their continental average and the total. The results of analyses of subsets of loci that were selected to have high gene diversities in either Europeans or Native Americans closely parallel those from the total set of loci. This suggests that the principal results are unlikely to be caused by a European ascertainment bias in locus selection. In summary, our findings demonstrate that partitions of gene diversity into within- and between-populations components are heavily biased by the populations analyzed and the models fitted. Optimistically, however, more information is available to analyze population history and evolution by quantifying, as we have done, the uniqueness of patterns of differentiation.