Organization and expression of canine olfactory receptor genes.

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.

Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.

The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.

Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.

Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.

Reproductive cooperation between queens and their mated workers: the complex life history of an ant with a valuable nest.

The life history of Harpegnathos saltator is exceptional among ants because both queens and workers reproduce sexually. Recently mated queens start new colonies alone, but later some of the offspring workers also become inseminated and take over the egg-laying role. This alternation seems associated with the existence of very complex underground nests, which are designed to survive floods. Longevity of ponerine queens is low (a consequence of limited caste dimorphism in this "primitive" subfamily), and upon the death of an H. saltator foundress, the nest represents a substantial investment. The queen's progeny should thus be strongly selected to retain the valuable nests. Unlike the flying queens, the workers copulate with males from their own colonies, and, thus, their offspring are expected to be highly related to the foundress. Colony fission appears not to occur because a daughter fragment would lack an adequate nest for protection. Thus, the annual production of queens in colonies with reproductive workers remains essential for the establishment of new colonies. This contrasts with various other ponerine species in which the queens no longer exist.

The genetic link between the Chinese bamboo partridge (Bambusicola thoracica) and the chicken and junglefowls of the genus Gallus.

Further comparison of mitochondrial control-region DNA base sequences of 16 avian species belonging to the subfamily Phasianinae revealed the following: (i) Generalized perdicine birds (quails and partridges) are of ancient lineages. Even the closest pair, the common quail (Coturnix coturnix japonica) and the Chinese bamboo partridge (Bambusicola thoracica), maintained only 85.71% identity. (ii) The 12 species of phasianine birds previously and presently studied belonged to three distinct branches. The first branch was made exclusively of members of the genus Gallus, while the second branch was made of pheasants of the genera Phasianus, Chrysolophus, and Syrmaticus. Gallopheasants of the genus Lophura were distant cousins to these pheasants. The great argus (Argusianus argus) and peafowls of the genus Pavo constituted the third branch. The position of peacock-pheasants of the genus Polyplectron in the third branch was similar to that of the genus Lophura in the second branch. Members of the fourth phasianine branch, such as tragopans and monals, were not included in the present study. (iii) The one perdicine species, Bambusicola thoracica, was more closely related to phasianine genera Gallus and Pavo than to members of other perdicine genera. The above might indicate that Bambusicola belong to one-stem perdicine lineage that later splits into two sublineages that yielded phasianine birds, one evolving to Gallus, and the other differentiating toward Pavo and its allies.

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.