Serotonin receptor 1A knockout: An animal model of anxiety-related disorder

To investigate the contribution of individual serotonin (5-hydroxytryptamine; 5-HT) receptors to mood control, we have used homologous recombination to generate mice lacking specific serotonergic receptor subtypes. In the present report, we demonstrate that mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety.

A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

Excess “read-through” acetylcholinesterase attenuates but the “synaptic” variant intensifies neurodeterioration correlates

Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The “read-through” variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the “synaptic” AChE variant, AChE-S. Several observations demonstrate that excess AChE-R attenuates, whereas AChE-S intensifies, neurodeterioration. In the somatosensory cortex, AChE-S transgenics, but not AChE-R or control FVB/N mice, displayed a high density of curled neuronal processes indicative of hyperexcitation. In the hippocampus, AChE-S and control mice, but not AChE-R transgenics, presented progressive accumulation of clustered, heat shock protein 70–immunopositive neuronal fragments and displayed a high incidence of reactive astrocytes. Our findings suggest that AChE-R serves as a modulator that may play a role in preventing the shift from transient, acute stress to progressive neurological disease.

An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases β-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes β-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene β-cyclase, an enzyme that converts lycopene to β-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that β-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalamus and with mutations at the db locus.

The gene product of the recently cloned mouse obese gene (ob) is important in regulating adipose tissue mass. ob RNA is expressed specifically by mouse adipocytes in vivo in each of several different fat cell depots, including brown fat. ob RNA is also expressed in cultured 3T3-442A preadipocyte cells that have been induced to differentiate. Mice with lesions of the hypothalamus, as well as mice mutant at the db locus, express a 20-fold higher level of ob RNA in adipose tissue. These data suggest that both the db gene and the hypothalamus are downstream of the ob gene in the pathway that regulates adipose tissue mass and are consistent with previous experiments suggesting that the db locus encodes the ob receptor. In db/db and lesioned mice, quantitative differences in expression level of ob RNA correlated with adipocyte lipid content. The molecules that regulate expression level of the ob gene in adipocytes probably are important in determining body weight, as are the molecules that mediate the effects of ob at its site of action.