Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est) and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

Exclusion of DMRT1 as a candidate gene for canine SRY-negative XX sex reversal.

DMRT1, which encodes a zinc finger-like DNA binding motif, is a well-conserved gene that is involved in testis differentiation in a variety of mammalian and non-mammalian vertebrates. The objective of this study was to determine whether a DMRT1 microsatellite marker allele is associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal generated from an American Cocker spaniel founder. Ten affected dogs and their parents and grandparents were genotyped. Four alleles at this locus and five different genotypes were found in this pedigree. All affected dogs inherited this trait from the foundation sire of this colony. Thus, the disease-causing mutation should be identical by descent in all affected dogs. Six affected dogs were found to have genotypes at this locus that were different from those of the founder sire. These results indicate that DMRT1 is an unlikely candidate gene for SRY-negative XX sex reversal in this model.

GABA(A) receptor epsilon and theta subunits display unusual structural variation between species and are enriched in the rat locus ceruleus.

Previously, GABA(A) receptor epsilon and theta subunits have been identified only in human. Here, we describe properties of the epsilon and theta subunit genes from mouse and rat that reveal an unusually high level of divergence from their human homologs. In addition to a low level of amino acid sequence conservation ( approximately 70%), the rodent epsilon subunit cDNAs encode a unique Pro/Glx motif of approximately 400 residues within the N-terminal extracellular domain of the subunits. Transcripts of the rat epsilon subunit were detected in brain and heart, whereas the mouse theta subunit mRNA was detectable in brain, lung, and spleen by Northern blot analysis. In situ hybridization revealed a particularly strong signal for both subunit mRNAs in rat locus ceruleus in which expression was detectable from the first postnatal day. Lower levels of coexpression were also detected in other brainstem nuclei and in the hypothalamus. However, the expression pattern of theta subunit mRNA was more widespread than that of epsilon subunit, being found also in the cerebral cortex of rat pups. In contrast to primate brain, neither subunit was expressed in the hippocampus or substantia nigra. The results indicate that GABA(A) receptor epsilon and theta subunits are evolving at a much faster rate than other known GABA(A) receptor subunits and that their expression patterns and functional properties may differ significantly between species.

Assessment of human gene diversity and expression patterns using expressed sequence tags.

ESTs provide a cheap and effective short-cut for identifying the majority of an organism's genes. Surveys of ESTs can provide a global view of the gene-expression patterns that distinguish different tissues or cell types. The large collection of human ESTs will soon permit the majority of genes to be placed on a chromosomal map.

cDNA sequencing: a means of understanding cellular physiology.

High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Modification of the GABA/benzodiazepine receptor with the arginine reagent, 2,3-butanedione.

Treatment of either crude or purified preparations of the gamma-aminobutyrate (GABA)/benzodiazepine receptor complex with arginine-specific reagents resulted in a time- and concentration-dependent loss of [3H]muscimol binding activity. Following exposure to either 2,3-butanedione or phenylglyoxal (less than or equal to 20 mM), [3H]muscimol binding was inhibited by up to 80%. [3H]Flunitrazepam binding was much less sensitive to the effects of the reagents. Scatchard analysis of the binding data indicated that treatment with butanedione resulted in a loss of [3H]muscimol binding sites with little effect on binding affinity. Considerable protection against inactivation was provided by arginine and by the endogenous receptor ligand, GABA. These results indicate that arginine residues play a critical role in maintaining the GABA receptor in a conformation capable of ligand binding, possibly by participating in the binding site through interaction with the carboxylate moiety of GABA.

5-HT(3)-receptor subunits A and B are co-expressed in neurons of the dorsal root ganglion.

The type 3 serotonin (5-HT(3)) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). Many pharmacological, behavioral, and electrophysiological studies indicate heterogeneous properties for this receptor. Although the basis for this heterogeneity is unknown, one possible explanation for these findings resides in the subunit composition of the receptor. Two 5-HT(3)-receptor subunits have been cloned: the 5-HT(3)-receptor subunit A (5-HT(3A)) and the 5-HT(3)-receptor subunit B (5-HT(3B)). Recombinant co-expression of 5-HT(3A) and 5-HT(3B) subunits produces a functional heteromeric 5-HT(3A/3B) receptor with pharmacological and electrophysiological properties different from those displayed by the 5-HT(3A) homomeric receptor. In the present report, we used in situ hybridization histochemistry to demonstrate that the 5-HT(3B) subunit is expressed in rat dorsal root ganglion (DRG) neurons. We determined with cellular resolution that 5-HT(3B) subunit mRNA was expressed in 43.2 +/- 2.8% of the total population of DRG neurons. By comparison, the 5-HT(3A) subunit was more widely expressed, with 70.0 +/- 2.8% of the total population of DRG neurons expressing this subunit. Further analyses showed that most of the neurons containing mRNA for the 5-HT(3B) subunit (91.5 +/- 3.4%) also expressed the 5-HT(3A) subunit. In contrast, nearly half the population of neurons expressing 5-HT(3A) subunit lacked (52.8 +/- 5.9%) transcripts for the 5-HT(3B) subunit. These results provide the first evidence indicating that the 5-HT(3B) subunit of the 5-HT(3) receptor is expressed in DRG and suggest that sensory neurons have the capacity to synthesize at least two structurally different 5-HT(3) receptors: a heteromeric 5-HT(3A/3B) receptor and a homomeric 5-HT(3A) receptor. Consequently, 5-HT(3) receptors with different properties might be present in peripheral and central axons of the DRG. These findings open the possibility that distinct types of 5-HT(3) receptors may be involved in perception and/or processing of sensory information. J. Comp. Neurol. 438:163-172, 2001. Published 2001 Wiley-Liss, Inc.

Introduction of the 5-HT3B subunit alters the functional properties of 5-HT3 receptors native to neuroblastoma cells.

The identification of a second 5-HT(3) (5-HT(3B)) subunit provides an explanation for 5-HT(3) receptor heterogeneity. We investigated whether introduction of recombinant 5-HT(3B) subunits would alter the functional properties of mouse neuroblastoma 5-HT(3) receptors. RT-PCR analysis revealed that NB41A3 cells contain mRNAs encoding 5-HT(3A) and 5-HT(3B) subunits. 5-HT increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and caused the concentration-dependent activation of inward currents recorded at -60 mV. Both actions of 5-HT were antagonized by ondansetron. The 5-HT concentration-response relationship of NB41A3 cells was indistinguishable from that of the related NG108-15 cell line. The selective 5-HT(3)-receptor agonist mCPBG also elevated [Ca(2+)](i) and activated inward currents. 2-M-5HT was less efficacious than 5-HT as an activator of 5-HT(3) receptors in NB41A3 cells and did not significantly increase [Ca(2+)](i). The 5-HT induced increase in [Ca(2+)](i) did not involve caffeine- or thapsigargin-sensitive intracellular Ca(2+) stores. The introduction of the 5-HT(3B) subunit by transient transfection of NB41A3 cells caused 5-HT to become less potent as an activator of 5-HT(3) receptors and altered the kinetics of 5-HT activated currents so that they resembled currents mediated by 5-HT(3AB) receptors. The 5-HT(3B) subunit also abolished the 5-HT induced [Ca(2+)](i) increase seen in untransfected NB41A3 cells. These data are consistent with the hypothesis that NB41A3 cells predominantly express homomeric 5-HT(3A) receptors that become heteromeric 5-HT(3AB) receptors upon introduction of the recombinant 5-HT(3B) subunit.

Alternative transcripts of the GABA(A) receptor epsilon subunit in human and rat.

In both human and rat tissues, complex patterns of transcripts are derived from the genes that encode the gamma-aminobutyric acid (GABA)(A) receptor epsilon subunit. An epsilon subunit transcript (approximately 3.6 kb) is expressed at relatively high levels in regions of the human brain and heart, but is not detected in most other major tissues. The encoded human epsilon subunit (epsilon (h)) confers distinctive properties to receptors into which it assembles. A distinct transcript of the gene (6.2 kb) is expressed abundantly in a variety of human tissues. This alternative transcript (ET2) appears to originate from within the epsilon subunit gene. It is possible that this transcript encodes a truncated subunit (epsilon (hS)), containing all of the transmembrane and intracellular domains. However, a combination of biochemical and electrophysiological analyses does not support this hypothesis. A distinct transcript of the epsilon subunit gene, encoding a large extracellular pro/glx domain, is expressed abundantly in rat and mouse brain. Functional analyses also failed to provide evidence for incorporation of this subunit (epsilon (rL)) into recombinant receptors. However, a shorter rat epsilon subunit (epsilon (r)), which lacks the pro/glx domain, conferred epsilon (h)-like properties to recombinant receptors, providing evidence for a functional rat epsilon subunit. In common with its human orthologue, incorporation of the epsilon (r) subunit into recombinant GABA(A) receptors confers several distinctive properties, including a reduced modulation by the anesthetic propofol and the appearance of spontaneous current.

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.

1. Radioligand binding and patch-clamp techniques were used to study the actions of gamma-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregnan-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABAA receptor subunits, expressed either alone or in combination. 2. Membranes from HEK293 cells after transfection with alpha 1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (< 0.03 pmol mg-1 protein). GABA (100 microM) applied to whole-cells transfected with alpha 1 cDNA and clamped at -60 mV, also failed to activate discernible currents. 3. The membranes of cells expressing beta 3 cDNAs bound [35S]-TBPS (approximately 1 pmol mg-1 protein). However, the binding was not influenced by GABA (10 nM-100 microM). Neither GABA (100 microM) nor picrotoxin (10 microM) affected currents recorded from cells expressing beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABAA receptors or spontaneously active ion channels. 4. GABA (10 nM-100 microM) modulated [35S]-TBPS binding to the membranes of cells transfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 microM-1 mM) also dose-dependently activated inward currents with an EC50 of 9 microM recorded from cells transfected with alpha 1 and beta 3 cDNAs, clamped at -60 mV. 5. Propofol (10 nM-100 microM), pentobarbitone (10 nM-100 microM) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 microM) modulated [35S]-TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 microM), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) also activated currents recorded from cells expressing alpha 1 beta 3 receptors. 6. Propofol (1 microM-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) failed to activate detectable currents. 7. Propofol (100 microM)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34 +/- 13% and 39 +/- 10% of control, respectively, by picrotoxin (10 microM). 5 alpha-Pregnan-3 alpha-ol-20-one (100 nM) enhanced propofol (100 microM)-evoked currents mediated by alpha 1 beta 3 receptors to 1101 +/- 299% of control. In contrast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) caused only a modest facilitation (to 128 +/- 12% of control) of propofol (100 microM)-evoked currents mediated by beta 3 homomers. 8. Propofol (3-100 microM) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 microM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a "surge' current on cessation of propofol (300 microM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) to cells expressing alpha 1 beta 3 receptors. 9. These observations demonstrate that rat beta 3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.

1. The modulation of the gamma-aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage-clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]-muscimol to a preparation of porcine brain membranes. 2. The progesterone metabolites 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one (greater than or equal to 30 nM) reversibly and dose-dependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 microM), and over the concentration range 30 nM-100 microM stimulated the binding of [3H]-muscimol. In contrast, 5 alpha- and 5 beta-pregnan-3 beta-ol-20-one (30 nM-100 microM) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3. Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]-muscimol binding sites as the principle action of the active steroid isomers. 4. GABA-evoked currents were also potentiated by androsterone (1 microM) and the deoxycorticosterone metabolite 5 alpha-pregnane-3 alpha,21-diol-20-one (100 nM). 5. Secobarbitone (10-100 microM), pentobarbitone (10-300 microM) and phenobarbitone (100-500 microM) reversibly and dose-dependently potentiated the amplitude of GABA-evoked currents in the absence of any change in their reversal potential. 6. At relatively high concentrations (greater than or equal to 30 microM) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor-channel activation since they share a common reversal potential with GABA-evoked responses (approximately 0 mV), are reversibly antagonized by bicuculline (3 microM), and potentiated by either diazepam (1 microM) or 5 beta-pregnan-3 alpha-ol-20-one (500 nM). 7. Secobarbitone (1 microM-1 mM) dose-dependently enhanced the binding of [3H]-muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8. A saturating concentration (1 mM) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5 beta-pregnan-3 alpha-ol-20-one (30 nM-100 microM). Similarly the steroid, at a concentration of 100 microM, did not influence the enhancement of [3H]-muscimol binding by secobarbitone (1 microM-1 mM). In all combinations of concentrations tested, the effects of secobarbitone and 5#-pregnan-3a-ol-20-one on [3H]-muscimol binding were additive. 9. In conjunction with previously published observations, the present data indicate close similarities in the GABA-mimetic and potentiating actions of barbiturates and steroids. However, the results obtained with combinations of steroids and barbiturates in the ligand binding assay appear inconsistent with the two classes of compound interacting with a common site to modulate the GABAA receptor activity.

The stimulatory effects of secobarbital and pregnanolone on the GABAA receptor can be blocked selectively.

The stimulatory effects of secobarbital and pregnanolone on GABA receptor binding could be distinguished by their sensitivity to blockade by phenobarbital, avermectin B1a and picrotoxinin. Phenobarbital (1 mM) and avermectin (10(-9)-10(-6) M) inhibited the stimulation of [3H]muscimol binding to pig brain membranes caused by secobarbital but not that caused by pregnanolone. In contrast, enhancement of [3H]muscimol binding by pregnanolone showed a greater sensitivity to the inhibitory effects of picrotoxinin. These results, together with data demonstrating an additivity of barbiturate- and steroid-induced effects, indicate that these two classes of modulators may interact with the GABAA receptor through different sites.

Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Characterization of a cytosolic protein (P36) isolated from pig brain by benzodiazepine-affinity chromatography.

Benzodiazepine-affinity chromatography, on a column of 1012S-Sepharose, resulted in the detection and purification of a binding protein (P36) from the cytosolic fraction of pig cerebral cortex. Purified P36 was enriched over 3,500-fold in a single step and was recovered with an efficiency of 50-60%. Analysis of the purified preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated a single polypeptide of Mr 36,000. The Stokes radius (3.44 nm) and sedimentation coefficient (4.43S) indicated that purified P36 is a dimeric protein. Analysis of the amino acid composition of P36 revealed a relatively high content of the hydrophobic amino acids, valine and leucine. Immunoblotting of several pig tissue preparations with an antiserum raised against purified P36 demonstrated approximately equal enrichment of P36 in cerebral cortex, cerebellum, and adrenal glands. Lesser enrichment was observed in kidney and liver, whereas a number of other tissues displayed no immunoreactivity. The gamma-aminobutyrate/benzodiazepine receptor complex and P36 showed no immunological cross-reactivity. High-affinity binding activity for [3H]Ro 15-4513, [3H]flunitrazepam, or [3H]PK11195 was not detected in preparations of purified P36. However, the ability of the gamma-aminobutyrate/benzodiazepine receptor inverse agonists, methyl- and ethyl-beta-carboline-3-carboxylate, to inhibit the binding of P36 to 1012S-Sepharose at relatively low concentrations indicates that P36 exhibits a degree of binding specificity.