Author Report for: Johnson CD

Wild populations of northern bobwhites (Colinus virginianus; hereafter bobwhite) have declined across nearly all of their U.S. range, and despite their importance as an experimental wildlife model for ecotoxicology studies, no bobwhite draft genome assembly currently exists. Herein, we present a bobwhite draft de novo genome assembly with annotation, comparative analyses including genome-wide analyses of divergence with the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genomes, and coalescent modeling to reconstruct the demographic history of the bobwhite for comparison to other birds currently in decline (i.e., scarlet macaw; Ara macao). More than 90% of the assembled bobwhite genome was captured within <40,000 final scaffolds (N50 = 45.4 Kb) despite evidence for approximately 3.22 heterozygous polymorphisms per Kb, and three annotation analyses produced evidence for >14,000 unique genes and proteins. Bobwhite analyses of divergence with the chicken and zebra finch genomes revealed many extremely conserved gene sequences, and evidence for lineage-specific divergence of noncoding regions. Coalescent models for reconstructing the demographic history of the bobwhite and the scarlet macaw provided evidence for population bottlenecks which were temporally coincident with human colonization of the New World, the late Pleistocene collapse of the megafauna, and the last glacial maximum. Demographic trends predicted for the bobwhite and the scarlet macaw also were concordant with how opposing natural selection strategies (i.e., skewness in the r-/K-selection continuum) would be expected to shape genome diversity and the effective population sizes in these species, which is directly relevant to future conservation efforts.

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.

We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.

Choline acetyltransferase (ChAT; EC 2.3.1.6) and acetylcholinesterase (AChE; EC 3.1.1.7) activities were measured in cynomolgus monkey ciliary muscle 1 month and 6 or more months after ciliary ganglionectomy (CG) or post-ganglionic ciliary neurectomy (PCN). ChAT activity was undetectable and AChE activity was elevated 1 month after CG or PCN, while both averaged about 30% of normal levels 6 or more months after denervation. Four out of six eyes reinnervated by functional criteria 6-12 months after CG or PCN. In one of the two remaining eyes permanently denervated, ChAT was absent from the ciliary muscle. In the other, ChAT activity was about 50% of normal, similar to the reinnervated eyes, but the regenerated cholinergic nerves were not approximated to the ciliary muscle fibers.

In C. elegans, the newly identified ace-3 is the third gene affecting acetylcholinesterase (AChE) activity. ace-3 II specifically affects class C AChE and is unlinked to ace-1 X or ace-2 I, which affect the other two AChE classes (A and B, respectively). Strains homozygous for an ace-3 mutation have no apparent behavioral or developmental defect; ace-1 ace-3 and ace-2 ace-3 double mutants are also nearly wild type. In contrast, ace-1 ace-2 ace-3 triple mutant animals are paralyzed and developmentally arrested; their embryonic development is relatively unimpaired, but they are unable to grow beyond the hatching stage. Based on the analysis of genetic mosaics, we conclude that in the absence of ace-2 and ace-3 function, the expression of ace-1(+) in muscle cells, but not in neurons, is essential for postembryonic viability.

Extracts of the nematode Caenorhabditis elegans contain five molecular forms of acetylcholinesterase (AChE) activity that can be separated by a combination of selective solubilization, velocity sedimentation, and ion-exchange chromatography. These are called form IA (5.2s), form IB (4.9s), form II (6.7s), form III (11.3s), and form IV (13.0s). All except form III are present in significant amounts in rapidly prepared extracts and are probably native; form III is probably derived autolytically from form IV. Most of forms IA and IB can be solubilized by repeated extractions without detergent, whereas forms II, III, and IV require detergent for effective solubilization and may therefore be membrane-bound. High salt concentrations are not required for, and do not aid in, the solubilization of these forms. For all forms, molecular weights and frictional ratios have been estimated by a combination of gel permeation chromatography and velocity sedimentations in both H2O and D2O. The molecular weight estimates range from 83,000 to 357,000 and only form II shows extensive asymmetry. The separated forms have been characterized with respect to substrate affinity, substrate specificity, inhibitor sensitivity, thermal inactivation, and detergent sensitivity. Judging by these properties, C. elegans is like other invertebrates in that none of its cholinesterase forms resembles either the "true" or the "pseudo" cholinesterase of vertebrates. However, internal comparison of the C. elegans forms clearly distinguishes forms IA, III, and IV as a group from forms IB and II; the former are therefore designated "class A" forms, the latter "class B" forms. Genetic evidence indicates that separate genes control class A and class B forms, and that these two classes overlap functionally. Several factors, including kinetic properties, molecular asymmetry, molecular size, and solubility, all suggest that a molecular model of the multiple cholinesterase forms observed in vertebrate electric organs probably does not apply in C. elegans. Potential functional roles and subunit structures of the multiple AChE forms within each C. elegans class are discussed.

Within a set of five separable molecular forms of acetylcholinesterase found in the nematode Caenorhabditis elegans, previously reported differences in kinetic properties identify two classes, A and B, likely to be under separate genetic control. Using differences between these classes in sensitivity to inactivation by sodium deoxycholate, a screening procedure was devised to search for mutants affected only in class A forms. Among 171 previously isolated behavioral and morphological mutant strains examined by this procedure, one (PR946) proved to be of the expected type, exhibiting a selective deficiency of class A acetylcholinesterase forms. Although originally isolated because of its uncoordinated behavior, this strain was subsequently shown to harbor mutations in two genes; one in the previously identified gene unc-3, accounting for its behavior, and one in a newly identified gene, ace-1, accounting for its selective acetylcholinesterase deficiency. Derivatives homozygous only for the ace-1 mutation also lacked class A acetylcholinesterase forms, but were behaviorally and developmentally indistinguishable from wild type. The gene ace-1 has been mapped near the right end of the X chromosome. Gene dosage experiments suggest that it may be a structural gene for a component of class A acetylcholinesterase forms.