Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis

Spermiogenesis in Caenorhabditis elegans involves the conversion of spherical, sessile spermatids into bipolar, crawling spermatozoa. In males, spermiogenesis is induced by mating, while in hermaphrodites, spermiogenesis occurs before the first oocytes are fertilized. Alternatively, spermiogenesis can be induced in vitro by treatment with monensin triethanolamine, or pronase. Treatment with the calmodulin inhibitors, trifluoperazine, chlorpromazine, or W7, also induces spermiogenesis in vitro with a half maximal effect at 20 microM. Upon initial activation, spermatids extend long, thin spikes and undergo extensive cellular movements. Eventually, a single motile pseudopod forms through the restructuring of one or more of these spikes. These transient spikes can be prolonged in vitro by removing triethanolamine as soon as the spermatids first form spikes. Spermatids from spe-8 and spe-12 spermatogenesis-defective (spe) mutants activate in vivo with male but not hermaphrodite sperm activator. In vitro, the mutant spermatids arrest spermiogenesis at the spike stage when activated with pronase, but form normal spermatozoa if subsequently or initially treated with monensin or triethanolamine. We present a model of spermiogenesis in which the mutant defects and the action of the pharmacological agents are ordered relative to one another.     

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.     

Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis.

The accuracy of DNA-based prenatal diagnosis of Duchenne muscular dystrophy (DMD) was determined by study of 174 families. Only 60% of families had a living affected male, and 63% had history of a single affected male. Prenatal diagnosis was declined by 47% of mothers whose DNA studies predicted a carrier risk below 2%, and none have had affected sons. Fetal risk was estimated prospectively by linkage analysis using intragenic and flanking RFLPs and retrospectively using dystrophin cDNA analysis for families whose linkage estimates lacked precision. Diagnostic accuracy was determined by comparing predictions with 40 male pregnancy outcomes. On the basis of linkage analysis, we anticipated 3.2 DMD males and observed 3.0. Retrospective cDNA analysis identified deletions in 2 of these 3 males. The combined use of linkage and cDNA deletion analysis provided a highly accurate method for prenatal diagnosis of DMD.     

Characterization of the major immunogen in the excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.     

A single base mutation in the gene for type III collagen (COL3A1) converts glycine 847 to glutamic acid in a family with Ehlers-Danlos syndrome type IV. An unaffected family member is mosaic for the mutation

Summary Ehlers-Danlos syndrome type IV, an inherited connective tissue disease, is usually caused by mutations in the gene for type III collagen. Here, we describe a glycine to glutamic acid substitution in a patient with this syndrome. Previous studies had shown that fibroblasts from the patient, his mother and brother secreted a reduced amount of type III collagen and also produced an overmodified form of the protein that was preferentially retained intracellularly. Peptide mapping experiments indicated that the mutation was located within cyanogen bromide peptide 9. This was supported by chemical cleavage analysis and sequencing of cDNA encoding this region. Allele-specific oligonucleotide hybridisation of genomic DNA confirmed that a G to A mutation converted Gly 847 to Glu. The mutation was present in two other affected family members and also in a third, who was clinically unaffected. Further analysis of this unaffected individual revealed reduced mutant:normal ratios in DNA obtained from both blood and hair samples, showing that she was mosaic for the mutation.     

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.     

Development and testing of protocols for computer-aided design of peptide drugs, using oxytocin

Considerable clinical interest in neuropeptides and peptide hormones has stimulated recent research and development of peptide-based drugs. This process differs from most classical drug discovery procedures because peptide molecules have considerable inherent flexibility. In the present paper, to identify lowest energy and metastable conformers for drug design, and to develop protocols for such studies, conformational search algorithms, incorporating empirical energy calculations, have been applied in the analysis of the peptide oxytocin. Energy minimization in torsion angle space was carried out from a variety of starting conformations, including published structures, in all-atom mode and all with distance constraints for disulphide bond formation. The energy-minimized conformations have been further optimized by a mapping method. Complementary simulations have been performed in united-atom mode and a model representing the effects of water using dummy sites has been developed and tested for this representation. Several of the preferred conformers together with de novo conformations have been used as starting points in molecular dynamics simulations; 28 low potential energy conformations were located at a temperature of 4 K. Conformations are analysed to identify hydrogen bonds, phi-psi angle distributions and the RMS values relative to the X-ray structure of deamino-oxytocin. The modelled structure of lowest energy in the molecular mechanics calculations was also that of least RMS deviation from the crystal structure; whilst structures of lower energy but larger deviation were identified by molecular dynamics techniques. A metastable structure has been identified which satisfies existing criteria for the "active form", and this model is tested by a theoretical residue-substitution technique, to provide clues on the agonist/antagonist relationship at the atomic level.     

The UL20 gene of herpes simplex virus 1 encodes a function necessary for viral egress.

A recombinant virus from which the start codon and 53% of the UL20 open reading frame had been deleted was constructed and characterized. We report the following: (i) The UL20- mutant formed small plaques in 143 tk- cells but failed to form plaques in Vero cells. Virus yields were approximately 10- to 100-fold lower than those of wild-type virus in all cell lines tested. (ii) Electron microscopic examination of Vero cells infected with the UL20- mutant revealed that enveloped and unenveloped capsids accumulated in the cytoplasm, possibly in the space between the inner and outer lamellae of the nuclear membrane, and that virtually no virus was present in the extracellular space. (iii) Glycoproteins B, C, D, E, H, and I recovered from lysates of cells infected with the UL20- mutant could not be differentiated from those present in lysates of cells infected with the wild-type parent virus with respect to the electrophoretic mobility of mature and precursor forms. (iv) Repair of the deleted sequences restored the wild-type phenotype. (v) The gene product of the UL20 gene was shown to be associated with cellular membranes and to possess characteristics of integral membrane proteins. We conclude that the UL20 gene encodes an integral membrane protein with a hitherto unrecognized function in that it enables the transit of virions to the extracellular space. The function of the UL20 gene product is complemented by some cell lines but not by Vero cells. The vesicles which serve to transport virions may have an origin different from those associated with transport of normal cellular proteins.     

Size-dependent drift responses of mayflies to experimental hydrologic variation: active predator avoidance or passive hydrodynamic displacement?

Summary Larger nymphs within aquatic insect taxa have been frequently observed to be transported down-stream in the stream drift only at night. Others have hypothesized this pattern results primarily from large nymphs' behavioural avoidance of entering drift during daylight, when size-selective, visually-feeding fish predators are most active. This hypothesis assumes that animals can actively control their entry into the drift, which may not be the case under all flow conditions. We experimentally induced streamflow increases and decreases in adjacent riffles in a hydrologically-stable stream during the daytime to examine whether changes in diel patterns of drift abundance and size-distribution of mayflies were consistent with the hypothesis of active avoidance of diurnal drift. We assessed the likelihood of active vs. passive mechanisms of diurnal drift entry and transport for four taxa that differ with respect to body size, morpho-behavioural attributes, microhabitat use, and general propensity to drift. In each of three seasons, diurnal and nocturnal drift samples were collected in three riffles over two diel cycles. Background drift patterns were established on the first day (no flow manipulation). Six h before sunset on the second day, flow was experimentally increased in one riffle, decreased in the second, and not altered in the third (control). Between-day differences in diurnal and nocturnal drift rate and size composition were then compared among the treatment and reference riffles. Responses of two taxa were consistent with active control over drift entry, transport, or both. For Baetis spp., drift-prone mayflies typically preyed upon by fish, diurnal drift rates immediately increased following both flow reduction and flow elevation in all seasons, but only small individuals comprised the drift. Drift by large individuals was delayed until nighttime. Epeorus longimanus also exhibited significant increases in drift rates following flow reduction and elevation, but responses of this large-bodied species were restricted to nighttime. Drift responses for these two taxa were largely independent of direction of hydrologic change, thus indicating a strong behavioural control over drift. By contrast, numbers and sizes of drifting Paraleptophlebia heteronea and Ephemerella infrequens depended strongly on direction of flow change. Drift rates for both species generally declined after flow reduction and increased after flow elevation. Moreover, after flow elevation, larger individuals often drifted diurnally, a finding consistent with expectations under a passive hydrodynamic model. These experiments indicate that size-dependent mayfly drift reflects not only presumed risk from visual fish predators, but also functional attributes of species such as morphology, behaviour, and microhabitat affiliation, which influence aspects of drift entry and transport under variable hydrologic conditions.