Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.     

A “Love” Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails

Animals have evolved many ways to enhance their own reproductive success. One bizarre sexual ritual is the “love” dart shooting of helicid snails, which has courted many theories regarding its precise function. Acting as a hypodermic needle, the dart transfers an allohormone that increases paternity success. Its precise physiological mechanism of action within the recipient snail is to close off the entrance to the sperm digestion organ via a contraction of the copulatory canal, thereby delaying the digestion of most donated sperm. In this study, we used the common garden snail Cornu aspersum to identify the allohormone that is responsible for this physiological change in the female system of this simultaneous hermaphrodite. The love dart allohormone (LDA) was isolated from extracts derived from mucous glands that coat the dart before it is stabbed through the partner''s body wall. We isolated LDA from extracts using bioassay-guided contractility measurement of the copulatory canal. LDA is encoded within a 235-amino acid precursor protein containing multiple cleavage sites that, when cleaved, releases multiple bioactive peptides. Synthetic LDA also stimulated copulatory canal contractility. Combined with our finding that the protein amino acid sequence resembles previously described molluscan buccalin precursors, this indicates that LDA is partially conserved in helicid snails and less in other molluscan species. In summary, our study provides the full identification of an allohormone that is hypodermically injected via a love dart. More importantly, our findings have important consequences for understanding reproductive biology and the evolution of alternative reproductive strategies.     

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Iota-carrageenan: molecular structure and packing of polysaccharide double helices in oriented fibres of divalent cation salts

The Ca²⁺ and Sr²⁺ salts of i-carrageenan have isomorphous crystal structures with trigonal unit cells of dimensions a = b = 1.373 nm, c = 1.328 nm, γ = 120 °. Two kinds of fibre diffraction pattern were found for the Mg²⁺ salt: one resembling the Ca²⁺ and Sr²⁺ patterns and one with additional layer lines interleaved midway between those in the usual kind of pattern. Specimens of this second type convert to the first type on storage at 92% relative humidity. These Mg²⁺i-carrageenate diffraction patterns provide direct evidence for the double-helical nature of the carrageenan molecule.A molecular model has been derived that consists of two, identical, righthanded, 3-fold helical polysaccharide chains of pitch 2.656 nm. One chain is translated axially 1.32 nm relative to the other.A packing arrangement with up-pointing and down-pointing double helices distributed randomly among the molecular sites explains the presence of both Bragg reflections and layer line streaks. The space group of our statistical crystal structure is P3₂12. The divalent cations were found by Fourier difference syntheses to be at ($\text{2}\text{3}$, $\text{1}\text{3}$, $\text{1}\text{6}$) and symmetry-related positions. The co-ordination of each cation to sulphate groups on two different helices leads to a continuous set of cation-sulphate-cation-… interactions that accounts for the high crystallinity of these salts.The structure of the Ca²⁺ salt has been refined by constrained linked-atom least-squares methods. The structural isomorphism of the Sr²⁺ salt was confirmed by an independent refinement.     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

Lipids and fatty acids in the copepod Jaschnovia brevis (Jaschnov) and in particulates from Arctic waters

The small, sub-ice copepod Jaschnovia brevis is rich in triacylglycerols, suggesting a feeding behaviour not constrained to the seasonal phytoplankton bloom. The copepod's triacylglycerol reserves contain: the diatom biomarkers 16:1n-7 (23.9%), 20:5n-3 (8.5%) and C16 PUFA (1.3%), the flagellate biomarkers 18:4n-3 (3.7%) and 22:6n-3 (3.3%), and the Calanus copepod biomarkers 20:1n-9 (7.7%) and 22:1n-11 (6.2%). Total lipid from particulates in the water column contained polar lipid (45.0%), wax esters (24.9%) and triacylglycerols (11.2%) as major components. The total lipids in the particulates were rich in 18:1n-9 (31.5%) and 16:0 (21.2%), and relatively rich in 18:0 (7.8%) and 18:2n-6 (9.2%). The triacylglycerols in the particulates contained 16:1n-7 (20.7%), C16 PUFA (4.1%), 18:4n-3 (1.9%), 20:5n-3 (3.6%), 22:6n-3 (1.9%), 20:1n-9 (5.2%) and 22:1n-11 (3.9%). The polar lipids in the particulates contained 16:1n-7 (17.3%), C16 PUFA (7.8%), 18:4n-3 (3.3%), 20:5n-3 (14.5%) and 22:6n-3 (9.6%). The fatty alcohols in the wax esters of the particulates were mainly 16:0 (11.3%), 20:1n-9 (21.1%) and 22:1n-11 (30.6%). The nature of the particulates, their possible origin in living and non-living material, and their role in the nutrition of J. brevis are discussed.     

Viability of glycerol-preserved and cryopreserved anuran skin

Summary Anurans are important animal models for studying the effects of anthropogenic chemical contamination of the environment. Two-compartment Teflon flow-through diffusion cells can be used to study percutaneus absorption of xenobiotics across harvested skin. However, such an approach currently necessitates that skin be harvested just before experimentation, a requirement that calls for the continuous growth and housing of living animals. The ability to preserve and store skin would allow more efficient use of animals and more flexibility in experimental design. To this end, we examined the viability of harvested anuran skin stored under various protocols consistent with current practices of mammalian skin preservation. Skin from the American bullfrog maintained 80–85% viability after 28 d, whereas viability of skin from the marine toad was only maintained for 7–10 d.