Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Mutations in QARS,Encoding Glutaminyl-tRNA Synthetase,Cause Progressive Microcephaly,Cerebral-Cerebellar Atrophy,and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

A sensitive microassay for protein in cells cultured on collagen

A microassay for protein that is linear from 0.1 to 5 μg of protein and does not detect collagen has been developed. The assay is based on the ability of bromosulphophthalein (BSP) to form BSP-protein complexes which precipitate at low pH. Maximum precipitation occurs when 30 or more BSP molecules are bound per albumin molecule. Collagen is not detected because too little BSP binds to this protein to precipitate it. This assay should be of great value to those who grow dispersed cell cultures on a collagen substrate.     

Transport of axonal enzymes in surviving segments of frog sciatic nerve

Redistribution of axonal enzymes as a function of time in vitro was studied in an unbranched segment of frog sciatic nerve. Cholinesterase activity moved peripherally at a rate of 99 mm/day and centrally at 19 mm/day. One-quarter of the total nerve content of the enzyme was estimated to be in motion, one-eighth in each direction. Mitochondrial enzymes (hexokinase and glutamic dehydrogenase) moved peripherally at 20–31 mm/day, centrally at 11–20 mm/day. Only 10% of the total content of these mitochondrial enzymes was in motion. No movement of choline acetylase or 6-phosphogluconic dehydrogenase activity was seen even after 4 days in vitro. However, in a 12 day in vivo experiment choline acetylase moved toward the periphery at a rate of 0.34 mm/day. After a day or so in vitro the distal accumulations of cholinesterase and glutamic dehydrogenase decreased, with a concomitant and quantitatively equivalent increase in enzyme activities at the proximal end of the nerve. It is postulated that during incubation a mechanism for reversing the direction of flow develops in the peripheral stump of the nerve. Vinblastine inhibited central and peripheral flow of both cholinesterase and glutamic dehydrogenase. Movement of cholinesterase was not affected by ouabain, thalidomide, or phenobarbital, nor by K⁺ excess (110 mM) or absence.     

Membrane marker movement on sympathetic axons in tissue culture

In cultures of dissociated chick sympathetic ganglia, retrograde (somatopetal) movement of concanavalin A receptors in the axolemma can be observed directly. Such movement was visualized by using concanavalin A coated red blood cells (ConA-RBCs) as membrane markers. Forty-eight percent of all ConA-RBCs which bound to sympathetic nerve fibers moved somatopetally at rates ranging from 11-84 μ/hr with a mean and standard error of 49 ± 6 μ/hr (n = 18). On nongrowing nerve fibers, the ConA- RBCs within 60 μ of the cell body showed retrograde movement, while on elongating neurites only those markers within 30 μ showed such movement. The rate of retrograde ConA-RBC movement appeared to increase with distance from the cell soma. The binding of ConA-RBCs to sympathetic neurites was specific for concanavalin A receptors since pretreatment with either concanavalin A or α-methylglucopyranoside prevented this binding. Untreated polystyrene beads (1.1 μ) which bound nonspecifically to the neurite membrane also showed retrograde movement. These beads moved somatopetally at rates similar to those of the ConA-RBCs but did so regardless of their initial distance from the soma. These data suggest that retrograde movement of surface elements might be a general property of cultured sympathetic nerve fibers.     

Effects of millimeter-wave radiation on monolayer cell cultures. I. Design and validation of a novel exposure system

A method has been devised whereby both the thermal and possible athermal biological effects resulting from microwave radiation can be assessed. Monolayer cultures of BHK-21/C13 cells were grown on microwave-transparent polystyrene coverslips, placed directly on the open end of a wave guide, and irradiated for 1 hour. In experiments seeking athermal biological effects of millimeter waves, culture medium was continuously recirculated over the cells to prevent temperature increases greater than 0.1 °C. Incorporation of ³H-uridine into RNA and of ³H-methionine into protein was quantified by measurement of optical densities of the autoradiographs in contiguous rectangular regions corresponding to portions of the cell monolayer immediately above the wave guide aperture and lying along its longer axis. Since power density was shown to vary with position along this axis according to a cosine² relationship, it was possible to assess the extent of microwave effects on macromolecular synthesis at power densities ranging from zero at each edge to twice the average power density at the center of the waveguide. Monolayer cultures maintained at 37.2 °C by recirculation of the medium did not show microwave-induced changes in synthesis of RNA and protein (41.8 or 74.0 GHz at average power densities of 320 or 450 mW/cm², respectively). Since macromolecular synthesis was examined both during and after irradiation, our results exclude both transient and persistent athermal biological effects of acute exposure to millimeter waves. In contrast, irradiation of cultures incubated in a small volume of nonrecirculated medium resulted in 1) marked heating of the monolayer, 2) a graded decline in macromolecular synthesis with increasing incident power, and 3), in some cases, destruction of the cell monolayer in the region immediately above the center of the waveguide aperture.