Expression of Xhdsi-1VOC, a novel member of the vicinal oxygen chelate (VOC) metalloenzyme superfamily, is up-regulated in leaves and roots during desiccation in the resurrection plant Xerophyta humilis (Bak) Dur and Schinz.

The annotation of novel plant genes is frequently based on sequence and structural similarity to known protein motifs. Understanding the biological function of these genes is dependent on identifying conditions under which they are activated, however. The resurrection plant, Xerophyta humilis is a good model system for identifying and characterizing genes which are important for desiccation tolerance. Desiccation induced-1 (dsi-1(VOC)), a previously uncharacterized plant gene, is up-regulated during desiccation in leaves, roots, and seeds in X. humilis. The X. humilis desiccation induced-1 gene, Xhdsi-1(VOC), shares structural homology with the vicinal oxygen chelate (VOC) metalloenzyme superfamily. Proteins in this superfamily share little sequence similarity, but are characterized by a common betaalphabetabetabeta structural fold. A number of plant orthologues of XhDsi-1(VOC) have been identified, including Arabidopsis thaliana At1g07645, which is currently annotated as a glyoxalase I-like gene, and many ESTs derived from seed cDNA libraries. Xhdsi-1(VOC) and its orthologues do not, however, contain the glutathione and zinc binding sites conserved in glyoxalase I genes. Furthermore, expression of Xhdsi-1(VOC) in yeast failed to rescue a yeast glyoxalase I mutant. Messenger RNA transcripts for At1g07645 accumulate during seed maturation, but are not induced by water loss, salt or mannitol stress in vegetative tissue in Arabidopsis. It is concluded that dsi-1(VOC) is a seed-specific gene in desiccation-sensitive plants that is activated by water loss in vegetative tissues in the resurrection plant X. humilis and plays an important role in allowing plant tissues to survive loss of 95% of their relative water content.

Development of GnRH cells: Setting the stage for puberty.

Cells containing gonadotropin-releasing hormone (GnRH) are essential not only for reproduction but also for neuromodulatory functions in the adult animal. A variety of studies have hinted at multiple origins for GnRH-containing cells in the developing embryo. We have shown, using zebrafish as a model system, that GnRH cells originate from precursors lying outside the olfactory placode: the region of the anterior pituitary gives rise to hypothalamic GnRH cells and the cranial neural crest gives rise to the GnRH cells of the terminal nerve and midbrain. Cells of both the forming anterior pituitary and cranial neural crest are closely apposed to the precursors of the olfactory epithelium during early development. Disruption of kallmann gene function results in loss of the hypothalamic but not the terminal nerve GnRH cells during early development. The GnRH proteins are expressed early in development and this expression is mirrored by the onset of GnRH receptor (GnRH-R) expression during early development. Thus the signaling of the GnRH neuronal circuitry is set up early in development laying the foundation for the GnRH network that is activated at puberty leading to reproductive function in the mature animal.

Candidate gene networks and blood biomarkers of methamphetamine-associated psychosis: an integrative RNA-sequencing report.

The clinical presentation, course and treatment of methamphetamine (METH)-associated psychosis (MAP) are similar to that observed in schizophrenia (SCZ) and subsequently MAP has been hypothesized as a pharmacological and environmental model of SCZ. However, several challenges currently exist in diagnosing MAP accurately at the molecular and neurocognitive level before the MAP model can contribute to the discovery of SCZ biomarkers. We directly assessed subcortical brain structural volumes and clinical parameters of MAP within the framework of an integrative genome-wide RNA-Seq blood transcriptome analysis of subjects diagnosed with MAP (N=10), METH dependency without psychosis (MA; N=10) and healthy controls (N=10). First, we identified discrete groups of co-expressed genes (that is, modules) and tested them for functional annotation and phenotypic relationships to brain structure volumes, life events and psychometric measurements. We discovered one MAP-associated module involved in ubiquitin-mediated proteolysis downregulation, enriched with 61 genes previously found implicated in psychosis and SCZ across independent blood and post-mortem brain studies using convergent functional genomic (CFG) evidence. This module demonstrated significant relationships with brain structure volumes including the anterior corpus callosum (CC) and the nucleus accumbens. Furthermore, a second MAP and psychoticism-associated module involved in circadian clock upregulation was also enriched with 39 CFG genes, further associated with the CC. Subsequently, a machine-learning analysis of differentially expressed genes identified single blood-based biomarkers able to differentiate controls from methamphetamine dependents with 87% accuracy and MAP from MA subjects with 95% accuracy. CFG evidence validated a significant proportion of these putative MAP biomarkers in independent studies including CLN3, FBP1, TBC1D2 and ZNF821 (RNA degradation), ELK3 and SINA3 (circadian clock) and PIGF and UHMK1 (ubiquitin-mediated proteolysis). Finally, focusing analysis on brain structure volumes revealed significantly lower bilateral hippocampal volumes in MAP subjects. Overall, these results suggest similar molecular and neurocognitive mechanisms underlying the pathophysiology of psychosis and SCZ regardless of substance abuse and provide preliminary evidence supporting the MAP paradigm as an exemplar for SCZ biomarker discovery.

Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis.

We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II>salmon GnRH>mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis.

Cloning and characterization of the chicken thyrotropin-releasing hormone receptor.

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.

Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function.

GnRH acts via GnRH receptors (GnRH-R) in the pituitary to cause the release of gonadotropins that regulate vertebrate reproduction. In the teleost fish, Haplochromis burtoni, reproduction is socially regulated through the hypothalamus-pituitary-gonadal axis, making the pituitary GnRH-R a likely site of action for this control. As a first step toward understanding the role of GnRH-R in the social control of reproduction, we cloned and sequenced candidate GnRH-R complementary DNAs from H. burtoni tissue. We isolated a complementary DNA that predicts a peptide encoding a G protein-coupled receptor that shows highest overall identity to other fish type I GnRH-R (goldfish IA and IB and African catfish). Functional testing of the expressed protein in vitro confirmed high affinity binding of multiple forms of GNRH: Localization of GnRH-R messenger RNA using RT-PCR revealed that it is widely distributed in the brain and retina as well as elsewhere in the body. Taken together, these data suggest that this H. burtoni GnRH receptor probably interacts in vivo with all three forms of GNRH:

Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail.

The chicken gonadotropin-releasing hormone receptor (GnRH-R) is notable for having a cytoplasmic C-terminal tail, which is not present in the mammalian GnRH-Rs. We report here that the cytoplasmic tail mediates rapid agonist-promoted receptor internalization. The chicken GnRH-R mediated internalization of gonadotropin-releasing hormone (GnRH) agonist (125I[His5-D-Tyr6]GnRH) at a rate of 11.3%.min-1, compared with only 0.71 %.min-1 for the human GnRH-R. To determine whether the presence of the cytoplasmic tail was responsible for the more rapid internalization kinetics of the chicken GnRH-R we truncated the tail after the Ile336 residue (S337stop). Receptor-mediated internalization of GnRH agonist by the S337stop-chicken GnRH-R was much slower than in the wild-type chicken receptor, and was similar to the wild-type human GnRH-R (0.55 %.min-1). These data indicate that rapid agonist-promoted internalization of the chicken GnRH-R is mediated through elements in the cytoplasmic C-terminal tail, distal to or including Ser337 and suggests that elimination of the C-terminal tail during evolution of mammalian GnRH-Rs may be related to its effects on internalization.

A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript.

Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.

Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen.

The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.

Cloning and characterization of the human GnRH receptor.

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

Distribution and regulation by oestrogen of fully processed and variant transcripts of gonadotropin releasing hormone I and gonadotropin releasing hormone receptor mRNAs in the male chicken.

The aim of this study was to increase understanding of the occurrence and regulation of chicken gonadotropin releasing hormone I (cGnRH I) and chicken gonadotropin releasing hormone receptor (cGnRH-R) mRNA variants in the hypothalamic-pituitary-testicular axis (HPTA). The study was carried out in the cockerel. Fully processed cGnRH I mRNA (cGnRH Ia) and a variant transcript (cGnRH Ib) with a retained intron 1 were observed in the preoptic/anterior hypothalamus (POA), the basal hypothalamus, anterior pituitary gland, and testes. Fully processed cGnRH-R mRNA (cGnRH-Ra) and a variant transcript (cGnRH-Rb) with a deletion were detected in the same tissues. In juvenile cockerels, concentrations of cGnRH Ia and b in the POA increased after castration, and this was prevented by oestrogen treatment. In the anterior pituitary gland, the concentration of cGnRH-Ra increased after castration and this was reversed by oestrogen treatment. In intact adult cockerels, oestrogen treatment depressed plasma luteinizing hormone but did not affect concentrations of cGnRH I and cGnRH-R mRNAs in the POA, basal hypothalamus, and anterior pituitary gland, suggesting that locally produced oestrogen, by aromatization, may exert maximal suppression on cGnRH I and GnRH-R mRNAs. In intact adult cockerels, the concentrations of cGnRH Ia and b in the testis, but not cGnRH-Ra and b, were depressed by oestrogen treatment. It was concluded that fully processed and variant cGnRH I and cGnRH-R mRNAs occur in all components of the HPTA. Oestrogen appears to play a role in the regulation of cGnRH Ia and b in the POA and testes, and of cGnRH-Ra in the POA and anterior pituitary gland.

Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector.

The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E. coli positive selection shuttle vector. The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E. coli and Streptomyces lividans respectively. The positive selection shuttle vector pLR591 facilitates the construction in E. coli of genomic libraries which can be screened in Streptomyces strains.

Genetic regulation of morphogenesis in Bacillus subtilis: roles of sigma E and sigma F in prespore engulfment.

Electron microscopic examination of sporulating cultures of wild-type Bacillus subtilis revealed that the morphological events previously characterized as stages II and III can be divided into four substages, namely, stages IIi, IIii, IIiii, and III. The ultrastructural phenotypes of several stage II mutant strains indicate that each of the four substages has a biochemical and genetic basis. Two of the genes needed for the transition from stage II to stage III encode transcription factors sigma E and sigma F. Their roles during spore morphogenesis have been the subject of much speculation. We now show that sigma E controls genes involved in the morphological transition from stage IIi to stage IIii and then stage IIiii, while the transition to stage III may be determined by genes controlled by sigma F. The results also indicate the existence of at least two undiscovered sporulation genes involved in B. subtilis spore morphogenesis.

The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother-cell-specific sporulation genes under the control of the sigma E form of RNA polymerase.

We have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter-proximal part of the spoIIIA locus. The locus consists of a polycistronic operon containing at least three genes. We show that the operon is regulated at the transcriptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation. Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation-specific sigma factor, sigma E. Moreover, synthesis of sigma E in vegetative cells by use of an inducible promoter causes expression of mother-cell-specific genes spoIID, spoIIIA, and spoIIID, but not the prespore-specific genes, spoIIIG and spoVA. We suggest that sigma E may be the primary determinant of mother-cell-specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor. Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient. Thus spoIIIA defines a third temporal class of gene controlled by the sigma E form of RNA polymerase.

The spoIIIA locus is not a major determinant of prespore-specific gene expression during sporulation in Bacillus subtilis.

During sporulation in Bacillus subtilis, expression of several prespore-specific genes is strongly dependent on the spoIIIE and spoIIIG gene products. Previous reports have also indicated a requirement for the products of the spoIIIA locus. However, we have now systematically studied six different well-defined spoIIIA mutations and find that, relative to spoIIIE and spoIIIG mutations, they have only a minor effect on the expression of two different prespore-specific genes, spoVA and sspA. Moreover, we have shown that strain IS37, which has been used as a spoIIIA mutant in several previous studies, actually contains a lesion in the spo0A gene. We conclude that spoIIIA has a relatively minor or indirect role in the regulation of prespore-specific gene expression.

Establishment of cell-specific transcription during sporulation in Bacillus subtilis.

One of the most intriguing questions posed by bacterial spore formation concerns the establishment of cell-specific gene expression in the prespore and mother cell. Recent results now suggest that sigma factors, in addition to their temporal roles in the control of gene expression, may also be the key determinants of differential gene expression during sporulation in Bacillus subtilis. The genes encoding two sporulation-specific sigma factors, sigma E and sigma F, are expressed soon after the initiation of sporulation, before the formation of the spore septum that separates the prespore and mother cell compartments. It now appears that sigma E and sigma F direct transcription only after septation and then in a specific cell type, suggesting that the segregation of the sigma activities after septation is a key event in the establishment of differential gene expression. The mechanism responsible for this segregation is complex, involving at least seven other gene products. We discuss possible models for the interactions between the sigma factors and the establishment of cell-specific transcription.

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after division, appear to be required only in the mother cell compartment.

Advances in understanding gonadotrophin-releasing hormone receptor structure and ligand interactions.

Gonadotrophin-releasing hormone (GnRH) is the central regulator of the reproductive system and its analogues are used widely in the treatment of diverse diseases. The GnRH receptor is a member of the large family of G-protein-coupled receptors (GPCRs) which have seven transmembrane domains. Knowledge of these receptors has assisted the development of molecular models of the GnRH receptor that allow prediction of its three-dimensional configuration and the way GnRH binds and activates its receptor. Comparison with other GPCRs led to the discovery that Lys121, in the third transmembrane domain, has a role in agonist binding. The history of GnRH structure-activity studies has allowed the identification of an acidic residue in the third extracellular loop of the receptor that is required for binding of mammalian GnRH, while synthetic GnRH analogues have showed that Asn102, in the second extracellular loop, may interact with the carboxy-terminus of GnRH. These residues can now be incorporated into the receptor models that are being used to design orally active non-peptide GnRH analogues for contraception and treatment of a variety of reproductive disorders.

SV-40 large T antigen reversibly inhibits expression of tyrosinase, TRP-1, TRP-2 and Mitf, but not Pax-3, in conditionally immortalized mouse melanocytes.

Transformation of mouse melanocytes with a variety of exogenous oncogenes or chemical carcinogens frequently results in irreversible loss of pigmentation. We have infected mouse melanocytes with a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen to study the molecular mechanisms underlying depigmentation during melanocyte transformation. The results show that, out of six cell lines analyzed at the permissive temperature of the oncoprotein, three epidermal and two dermal melanocyte clones remained pigmented and retained the ability to synthesize melanin and to express the melanocyte-specific genes, including tyrosinase, tyrosinase related protein-1, tyrosinase related protein-2 and Mitf. In contrast, one dermal melanocyte clone (DMEL-3) gradually depigmented. This depigmentation was characterized by enhanced growth and down-regulation of melanocyte-specific gene expression. When the oncogene was inactivated by culture at the non-permissive temperature, the pigmented phenotype in DMEL-3 cells could be rescued, and there was a corresponding time-dependent increase in melanocyte-specific gene expression. After extended passage, this rescue could not be achieved. Our results provide direct evidence for the role of the SV40 large T antigen in melanocyte de-differentiation. Expression of Pax-3, a transcription factor implicated in melanocyte differentiation, was unaltered during the SV40-initiated de-differentiation, and de-differentiated melanocytes expressed normal levels of Pax-3 message. We speculate on the mechanism by which the oncoprotein might be regulating Mitf gene expression and of the role of Pax-3 in this process.