Genetic evidence for two species of elephant in Africa.

Elephants from the tropical forests of Africa are morphologically distinct from savannah or bush elephants. Dart-biopsy samples from 195 free-ranging African elephants in 21 populations were examined for DNA sequence variation in four nuclear genes (1732 base pairs). Phylogenetic distinctions between African forest elephant and savannah elephant populations corresponded to 58% of the difference in the same genes between elephant genera Loxodonta (African) and Elephas (Asian). Large genetic distance, multiple genetically fixed nucleotide site differences, morphological and habitat distinctions, and extremely limited hybridization of gene flow between forest and savannah elephants support the recognition and conservation management of two African species: Loxodonta africana and Loxodonta cyclotis.

Cloning of a structural and functional homolog of the circadian clock gene period from the giant silkmoth Antheraea pernyi.

The period (per) gene of Drosophila plays an important role in circadian clock function. Interestingly, homologs of per have not been cloned outside of dipteran species. Using a PCR strategy, we now report the cloning of the cDNA of a per homolog from the silkmoth Antheraea pernyi. The cDNA encodes a protein of 849 amino acids, which shows highest identity (39%) with the per protein of Drosophila virilis. Stretches of high identity between moth and fly proteins are in the amino terminus, the PAS region, and the region surrounding the site of the per mutation in Drosophila. Moth per homolog mRNA levels exhibit a prominent circadian variation in adult heads, and per protein antibodies show a pronounced variation of per antigen staining in photoreceptor nuclei. With sequence information derived from moth and flies, per-like cDNA fragments were readily cloned by PCR from other moth species and a third insect order.

Period protein is necessary for circadian control of egg hatching behavior in the silkmoth Antheraea pernyi.

We examined the molecular basis of the circadian control of egg hatching behavior in the silkmoth Antheraea pernyi. Egg hatching is rhythmically gated, persists under constant darkness, and can be entrained by light by midembryogenesis. The time of appearance of photic entrainment by the silkmoth embryo coincides with the appearance of Period (PER) and Timeless (TIM) proteins in eight cells in embryonic brain. Although daily rhythms in PER and/or TIM immunoreactivity in embryonic brain were not detected, a robust circadian oscillation of PER immunoreactivity is present in the nuclei of midgut epithelium. per antisense oligodeoxynucleotide treatment of pharate larvae on the day before hatching consistently abolishes the circadian gate of egg hatching behavior. per antisense treatment also causes a dramatic decrease in PER immunoreactivity in newly hatched larvae. The results provide direct evidence that PER is a necessary element of a circadian system in the silkmoth.

Structure, characterization, and expression of the gene encoding the mouse Mel1a melatonin receptor.

Recently, a distinct family of G protein-coupled receptors has been cloned that mediates the biological effects of melatonin. Of two sub-types cloned from mammals (Mel1a and Mel1b), the Mel1a receptor appears to mediate the circadian and reproductive effects of the hormone. We now report the cloning, characterization, and expression of the gene encoding the Mel1a receptor in mice. The receptor gene is composed of two exons, separated by an intron of greater than 13 kilobases. Exon 1 encodes the entire 5'-untranslated region and the coding region through the first cytoplasmic loop. Exon 2 encodes the rest of the coding region and the entire 3'-untranslated region. 5'-Rapid amplification of complementary DNA ends and ribonuclease protection analyses show that the major transcription start site is 103 nucleotides upstream of the translation start codon. Sequence analysis of 1.1 kilobases of the 5'-flanking region reveals that it does not contain TATA or CAAT boxes. The 5'-flanking region drives luciferase expression 114-fold over basal levels in a murine retinal cell line that endogenously expresses the Mel1a receptor. The mouse receptor binds 2-[125]iodomelatonin with high affinity (K(d) = 55.6 pM) when expressed transiently in COS-7 cells. In situ hybridization studies establish that Mel1a receptor messenger RNA is expressed in the hypothalamic suprachiasmatic nuclei and hypophyseal pars tuberalis, presumed sites of the circadian and some of reproductive actions of melatonin, respectively. These results provide information on Mel1a receptor gene structure essential for designing transgenic and gene knock-out studies and analyzing the transcriptional regulation of receptor gene expression.

Comparative efficacy of expression of genes delivered to mouse sensory neurons with herpes virus vectors.

To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E. coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout. Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal). With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation. A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points. With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1. No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk. Immunocytochemistry for E. coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining. A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout. These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion.

Serotonin receptor gene expression in the rat suprachiasmatic nuclei.

Serotonin (5HT) is thought to reset the biological clock in the suprachiasmatic nuclei (SCN) in vitro through a postsynaptic 5HT-1a receptor. Thus we examined 5HT receptor gene expression in the SCN by in situ hybridization. On film autoradiograms, 5HT-1c receptor mRNA showed intense SCN hybridization, while 5HT-1b receptor mRNA displayed a weaker signal. Emulsion autoradiograms additionally revealed expression of 5HT-1a and 5HT-2 receptor mRNAs by a few scattered SCN cells. 5HT-3 receptor mRNA was not detected in the SCN, although the transcript was detected elsewhere in the brain. 5HT-1d and -1e receptor mRNAs were not detected in the SCN or elsewhere in brain within the sections examined. The results do not support a major role for postsynaptic 5HT-1a receptors in resetting SCN rhythms.

c-fos and jun-B mRNAs are transiently expressed in fetal rodent suprachiasmatic nucleus following dopaminergic stimulation.

We examined the time-course of expression of c-fos and jun-B mRNAs in the fetal rat suprachiasmatic nuclei (SCN) following maternal cocaine injection on gestational day 20. Both c-fos and jun-B mRNA levels increased within 20 min, peaked at 40 min and declined to baseline by 120 min after cocaine treatment (30 mg/kg). In mice, the D1-dopamine agonist, SKF 38393, induced c-fos and jun-B mRNAs in the fetal SCN and striatum. Regulated expression of immediate early genes in the fetal SCN may play a role in entrainment of the fetal clock.

Cellular organization in rat somatosensory cortex: effects of sex and laterality.

Layer IV of rodent somatosensory cortex contains distinct arrangements of cells characterized as barrels. When barrels first form in rats, each barrel consists of a cell-dense "wall" and a cell-sparse "hollow." With age, the distinction of the boundary between barrel walls and hollows diminishes. Cellular arrangements within barrels were quantified to test whether the barrels are influenced by sex and laterality during cortical development. A computer-assisted method was developed to measure cell densities in relation to barrel boundaries. The boundaries between barrel walls and hollows were determined in tissue double-stained for Nissl substance and cytochrome oxidase histochemistry. The distinction between barrel walls and hollows revealed by Nissl stains differed significantly between anterior and posterior barrels. This distinction declined significantly in anterior barrels from Postnatal Day 10 (P10) to P30. The area of cortex containing barrels was estimated from composites of Nissl-stained sections. At P20 the detectable barrel cortex area was larger on the right in females and on the left in males resulting in a significant sex difference in barrel cortex asymmetry. This sex difference in barrel cortex laterality was detected only in Nissl-stained tissue; there were no differences attributable to sex or side in barrel cortex area analyzed for cytochrome oxidase reactivity. We hypothesize that sex-dependent differences in barrel cortex structure result from lateralized differences in cellular organization.

Mapping of the gene for the Mel1a-melatonin receptor to human chromosome 4 (MTNR1A) and mouse chromosome 8 (Mtnr1a).

The pineal hormone melatonin elicits potent circadian and reproductive effects in mammals. We report the chromosomal location of the gene for the Mel1a-melatonin receptor that likely mediates these circadian and reproductive actions. PCR analysis of human-rodent somatic cell hybrids showed that the receptor gene (MTNR1A) maps to human chromosome 4q35.1. An interspecific backcross analysis revealed that the mouse gene (Mtnr1a) maps to the proximal portion of chromosome 8. These loci may be involved in genetically based circadian and neuroendocrine disorders.