Synergistic effects of genetic variation in nicotinic and muscarinic receptors on visual attention but not working memory.

It is widely appreciated that neurotransmission systems interact in their effects on human cognition, but those interactions have been little studied. We used genetics to investigate pharmacological evidence of synergisms in nicotinic/muscarinic interactions on cognition. We hypothesized that joint influences of nicotinic and muscarinic systems would be reflected in cognitive effects of normal variation in known SNPs in nicotinic (CHRNA4 rs1044396) and muscarinic (CHRM2 rs8191992) receptor genes. Exp. 1 used a task of cued visual search. The slope of the cue size/reaction time function showed a trend level effect of the muscarinic CHRM2 SNP, no effect of the nicotinic CHRNA4 SNP, but a significant interaction between the 2 SNPs. Slopes were steepest in individuals who were both CHRNA4 C/C and CHRM2 T/T homozygotes. To determine the specificity of this synergism, Exp. 2 assessed working memory for 1-3 locations over 3 s and found no significant effects on either SNP. Interpreting these results in light of Sarter's [Briand LA, et al. (2007) Modulators in concert for cognition: Modulator interactions in the prefrontal cortex. Prog Neurobiol 83:69-91] claims of tonic and phasic modes of cholinergic activity, we argue that reorienting attention to the target after invalid cues requires a phasic response, dependent on the nicotinic system, whereas orienting attention to valid cues requires a tonic response, dependent on the muscarinic system. Consistent with that, shifting and scaling after valid cues (tonic) were strongest in CHRNA4 C/C homozygotes who were also CHRM2 T/T homozygotes. This shows synergistic effects within the human cholinergic system.

An animal model for the molecular genetics of CADASIL. (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).

BACKGROUND: CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an inherited condition that causes repeated small-scale strokes in adults. CADASIL is caused only by mutations in the human NOTCH3 gene that increase or decrease the number of cysteines within the epidermal growth factor (EGF) repeats of the NOTCH3 protein. Drosophila: lethal-Abruptex is a similar condition because it is also caused only by mutations that increase or decrease the number of cysteines within the EGF repeat portion of the Notch protein. SUMMARY OF COMMENT: Drosophila: lethal-Abruptex and human CADASIL are precisely analogous at the molecular level, and both are genetically dominant. These precise similarities, together with the fact that the structure and function of Notch has been highly conserved throughout the animal kingdom, provide an animal model for the molecular and genetic aspects of human CADASIL. It also provides support for Spinner's proposal that CADASIL results from dominant inhibition of the Notch pathway. CONCLUSIONS: Because the phenotypes of Notch mutations are cell-autonomous, the symptoms of CADASIL indicate that adult vascular smooth muscle cells require the continuing function of the NOTCH3 pathway in the adult. For this reason, further analysis of the NOTCH3 pathway may provide more general insights into the biology of vascular smooth muscle cells. In the case of CADASIL, the powerful genetic tools available in Drosophila: should help to facilitate future research.

Cytosine deamination plays a primary role in the evolution of mammalian isochores.

DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals.

Pantropic retroviral vectors mediate somatic cell transformation and expression of foreign genes in dipteran insects.

The control of insects that transmit disease and damage crops has become increasingly difficult. The ability to genetically engineer insects would facilitate strategies to protect crops and block arthropod vector-borne disease transmission. Transformation vectors based on insect transposable elements have been developed, but most have limited host ranges. A promising alternative is the pantropic retroviral vector, which is packaged with the envelope glycoprotein from vesicular stomatitis virus and is replication-defective. We show here that pantropic murine retroviral vectors can mediate high-level expression of foreign genes in somatically transformed insect larvae and adults of three dipteran genera. This success demonstrates the potential for germline transformation mediated by pantropic retroviral vectors.

The coevolution of gene family trees.

Gene duplication mutants arise spontaneously at a high rate in bacteria, bacteriophages, insects and mammalian cells, and are generally viable. Thus, the rate-limiting step in the evolutionary process of gene duplication and divergence was probably not gene duplication per se. Rather, it is likely that only a small fraction of all duplicated genes were retained, and were able to diverge into new specificities. Furthermore, gene duplications and functionally related gene families often show similarities in divergence dates, functional specificities, and phylogenetic tree topologies. These correlations suggest that the family trees of functionally related gene families co-evolved because functionally complementary gene duplication and divergence events tended to be retained by natural selection.

The evolutionary divergence of neurotransmitter receptors and second-messenger pathways.

Members of the superfamily of G-protein-coupled neurotransmitter receptors have a conserved secondary structure, a moderate and reasonably steady rate of sequence change, and usually lack introns within the coding sequence. These properties are advantageous for evolutionary studies. The duplication and divergence of the genes in this gene family led to the formation of distinct neurotransmitter pathways and may have facilitated the evolution of complex nervous systems. I have analyzed this evolutionary divergence by quantitative multiple sequence alignment, bootstrap resampling, and statistical analysis of 49 adrenergic, muscarinic cholinergic, dopamine, and octopamine receptor sequences from 12 animal species. The results indicate that the first event to occur within this gene family was the divergence of the catecholamine receptors from the muscarinic acetylcholine receptors, which occurred prior to the divergence of the arthropod and vertebrate lineages. Subsequently, the ability to activate specific second-messenger pathways diverged independently in both the muscarinic and the catecholamine receptors. This appears to have occurred after the divergence of the arthropod and vertebrate lineages but before the divergence of the avian and mammalian lineages. However, the second-messenger pathways activated by adrenergic and dopamine receptors did not diverge independently. Rather, the ability of the catecholamine receptors to bind to specific ligands, such as epinephrine, norepinephrine, dopamine, or octopamine, was repeatedly modified in evolutionary history, and in some cases was modified after the divergence of the second-messenger pathways.

Genetic mosaic analysis of the equatorial-less mutation in Drosophila melanogaster.

eql (equatorial-less) is a recessive lethal mutation on the second chromosome of Drosophila melanogaster. J. Campos-Ortega found that eql clones in somatic mosaic flies have reduced numbers of photoreceptor cells, and he suggested that only the R1, R6, and R7 photoreceptor cells were missing in this mutant. These photoreceptor cells help to define the inverted orientation of ommatidial facets along the equatorial midline of the fly eye, hence the mutation was named "equatorial-less." We have conducted a detailed analysis of the eql mutation, by serial section reconstruction of eql clones marked with bw- or w- in somatic mosaic flies. We found that all photoreceptor cell types (R1-R8) could be deleted by the eql mutation, and in rare cases the number of photoreceptor cells was increased. The apparent lack of photoreceptor cell type specificity was confirmed by our analysis of genetically mosaic facets, which indicated that no single photoreceptor cell, or subset of photoreceptor cells, was uniquely required to express eql+. Rather, eql appears to function in all photoreceptor cells, and possibly in all eye precursor cells. The distribution of photoreceptor cell numbers in w eql facets was consistent with the hypothesis that each photoreceptor cell was deleted independently of the others. The eql gene is located on the right arm of chromosome 2 at map location 2-104.5 +/- 0.7 and lies between the polytene chromosome bands 59D8 and 60A7.

Characterization of the radiation-sensitive stage in the development of the compound eye of Drosophila.

The development of the compound eye of Drosophila is particularly sensitive to irradiation during the third larval instar. Moreover, the anterioposterior location of the eye-pattern defects produced by irradiation during the third instar is correlated with the age of the larvae at the time of irradiation, as first shown by H.J. Becker. The development of the fly eye proceeds from posterior to anterior, and so these results suggest that there may be a radiation-sensitive stage in the development of the precursor cells in the eye imaginal disc of Drosophila. We show here that irradiation of third-instar Drosophila larvae with 23-30 Gy of 60Co gamma-rays produces confluent pattern disruptions in a dorsoventral stripe of eye tissue with an average width of about 8 facets along the anterioposterior axis. By measuring the time interval from irradiation to pupariation in individual larvae, we were able to determine that the posterior boundary of the radiation-sensitive region is located 0-1 columns anterior to the morphogenetic furrow in the developing eye imaginal disc. Therefore the anterior boundary of the radiation-sensitive region lies about 8-9 columns anterior to the morphogenetic furrow. These boundaries demarcate the region of the eye imaginal disc within which a specific subset of precursor cells (those that will develop into the R1, R6 and R7 photoreceptor cells, as well as the pigment and cone cells) are preparing for their final round of mitosis. Irradiation of these precursor cells would cause the death or delayed mitosis of their daughter cells within the morphogenetic furrow, while they are initiating the cellular interactions that determine cell fate in the developing eye. Irradiation of more anterior cells (i.e., at earlier stages) results in few pattern defects, presumably because the resulting cell death and delayed mitosis can be completed before the morphogenetic furrow passes.

The evolution of rhodopsins and neurotransmitter receptors.

Rhodopsins share a limited number of amino acid identities with a variety of other integral membrane proteins. Most of these proteins have seven putative transmembrane segments and are likely to play a role in transmembrane signaling. We have undertaken a systematic series of comparisons of primary and secondary structure in order to clarify the functional and evolutionary significance of these sequence similarities. On the basis of consistently high similarity scores, we find that the most internally consistent definition of the rhodopsin gene family would include vertebrate rhodopsins, alpha- and beta-adrenergic receptors, M1 and M2 muscarinic acetylcholine receptors, substance K receptors, and insect rhodopsins, while excluding bacteriorhodopsin, the mass human oncogene, vertebrate and insect nicotinic acetylcholine receptors, and the yeast STE2 and STE3 peptide receptors. The rhodopsin gene family is highly diverged at the primary sequence level but has maintained a conserved secondary structure, including a previously unidentified hierarchy of transmembrane segment hydrophobicity. We have developed new computer algorithms for progressive multiple sequence alignment and the analysis of local conservation of protein domains, and we have used these algorithms to examine the phylogeny of the rhodopsin gene family and the changing domains of sequence conservation. The results show striking differences and similarities in the conserved domains in each of the three main branches of the rhodopsin gene family, and indicate that color vision arose independently in the lines of descent leading to modern humans and fruit flies.

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.

Development and applications of a solid-phase radioimmunoassay for the PO protein of peripheral myelin.

This is the first report of a quantitative radioimmunoassay for PO. The assay uses antigen-coated plastic microwells, with antibody binding detected by 125I-labeled protein A. Either peripheral myelin proteins or purified PO may be used as the antigen. Optimal extraction of tissue samples for PO immunoassay requires careful attention to the sodium dodecyl sulfate-to-protein ratio. Sodium dodecyl sulfate interference with antibody binding can be minimized by adding an excess of nonionic detergent and carrier protein to the incubation buffer. This method allows the detection of 0.8 ng of PO (20 ng/ml). Results from this assay showed little or no immunoreactivity in extracts of brain, centra myelin, liver, purified myelin basic proteins, cultured, purified secondary Schwann cells, or membrane preparations from these cells. PO was clearly detectable in Schwann cell cultures from 3- to 4-day-old rats at 12-18 h after dissociation (4% of the level in adult sciatic nerve) and in extracts of one-day-old rat sciatic nerve (2% of the level in adult nerve). Myelin basic protein radioimmunoassays showed that the ratio of PO to myelin basic protein is essentially constant in extracts of sciatic nerve from ne-day-old, four-day-old, and young adult rats. Another result was that PO levels are reduced in the trembler mouse sciatic nerve.

Studies on cultured Schwann cells: the induction of myelin synthesis, and the control of their proliferation by a new growth factor.

We have recently described the use of immunological methods to identify and purify rat Schwann cells. In dissociated cultures of neonatal sciatic nerve, all of the cells can be identified by antigenic criteria as either Schwann cells or fibroblasts. The fibroblasts may be removed by treatment with antiserum to the Thy-1 antigen and complement. The purified Schwann cells have been used to study the regulation of the expression of myelin components, and the stimulation of Schwann cell division by a soluble growth factor. Among the components of myelin, we have concentrated on the peripheral myelin glycoprotein P0, which constitutes 50-60% of the protein in peripheral myelin. We have studied the distribution of P0 in vitro and in vivo by immunofluorescence, immuno-autoradiography on SDS gels, and solid-phase radioimmunoassay. Our results support the hypothesis that P0 is induced specifically as a consequence of the interaction between the Schwann cell and the myelinated type of axon. The level of P0 in the myelin membrane is at least 1000-fold higher than in the Schwann cell membrane. Purified Schwann cells divide very slowly in a conventional tissue culture medium. This has allowed us to purify a new growth factor from extracts of brain and pituitary, tentatively named Glial Growth Factor (GGF). The activity resides in a basic protein with a native molecular weight of 6 x 10(4) daltons and a subunit molecular weight of 3 x 10(4) daltons, which is active at levels comparable to those of epidermal growth factor. GGF is mitogenic for Schwann cells, astrocytes and muscle fibroblasts.

Synthesis of sulfatide by cultured rat Schwann cells.

The 35S sulfolipids synthesized by purified cultures of rat Schwann cells, fibroblasts, and a rat cell line (RN2) were studied. Schwann cell 35S sulfolipids were almost entirely [35S]sulfatide, as shown by TLC in two different solvent systems with unlabeled authentic sulfatide run in the same track. RN2 and fibroblasts did not synthetize significant amounts of sulfatide, by the same criteria. Previous studies failed to detect any characteristic myelin components, including sulfatide, on Schwann cells after several days in culture )Brockes et al., 1980a; Mirsky et al., 1980). My results show that Schwann cells continue to synthesize some sulfatide in the absence of neurons.