Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of ≈100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5–10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Computerized polymorphic marker identification: Experimental validation and a predicted human polymorphism catalog

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called pompous (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by pompous to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. pompous provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use pompous to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction

Nuclear receptors regulate metabolic pathways in response to changes in the environment by appropriate alterations in gene expression of key metabolic enzymes. Here, a computational search approach based on iteratively built hidden Markov models of nuclear receptors was used to identify a human nuclear receptor, termed hPAR, that is expressed in liver and intestines. hPAR was found to be efficiently activated by pregnanes and by clinically used drugs including rifampicin, an antibiotic known to selectively induce human but not murine CYP3A expression. The CYP3A drug-metabolizing enzymes are expressed in gut and liver in response to environmental chemicals and clinically used drugs. Interestingly, hPAR is not activated by pregnenolone 16α-carbonitrile, which is a potent inducer of murine CYP3A genes and an activator of the mouse receptor PXR.1. Furthermore, hPAR was found to bind to and trans-activate through a conserved regulatory sequence present in human but not murine CYP3A genes. These results provide evidence that hPAR and PXR.1 may represent orthologous genes from different species that have evolved to regulate overlapping target genes in response to pharmacologically distinct CYP3A activators, and have potential implications for the in vitro identification of drug interactions important to humans.

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Identification of the proton pathway in bacterial reaction centers: Replacement of Asp-M17 and Asp-L210 with Asn reduces the proton transfer rate in the presence of Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule QB (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, kAB(2) [QA⨪QB⨪ + H+ → QA(QBH)−] in native and mutant RCs in the absence and presence of Cd2+. Previous work has shown that the binding of Cd2+ decreases kAB(2) in native RCs ≈100-fold. The preceding paper shows that bound Cd2+ binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with QB⨪ by searching for mutations that greatly affect kAB(2) (≳10-fold) in the presence of Cd2+, where kAB(2) is limited by the proton transfer rate (kH). Upon mutation of Asp-L210 or Asp-M17 to Asn, kH decreased from ≈60 s−1 to ≈7 s−1, which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (kH ≈ 7 s−1) with that in native RCs in the absence of Cd2+ (kH ≥ 104 s−1), we conclude that alternate proton transfer pathways, which have been postulated, are at least 103-fold less effective.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts

This paper describes three distinct estrogen receptor (ER) subtypes: ERα, ERβ, and a unique type, ERγ, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERγ arose through gene duplication from ERβ early in the teleost lineage and indicates that ERγ is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERγ shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERγs. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERβ and ERγ fusion proteins can each bind estradiol-17β with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-α, -β, or -γ treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (α, β) or Type II (γ) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1α). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by “mixed-peptide” Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.