Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif.

Elements responsible for dexamethasone responsiveness of CYP3A23, a major glucocorticoid-inducible member of the CYP3A gene family, have been identified. DNase I footprint analysis of the proximal promoter region revealed three protected sites (sites A, B, and C) within the sequence defined by -167 to -60. Mutational analysis demonstrated that both sites B and C were necessary for maximum glucocorticoid responsiveness and functioned in a cooperative manner. Interestingly, neither site contained a glucocorticoid responsive element. Embedded in site C was an imperfect direct repeat (5'-AACTCAAAGGAGGTCA-3'), showing homology to an AGGTCA steroid receptor motif, typically recognized by the estrogen receptor family, while site B contained an ATGAACT direct repeat; these core sequences were designated dexamethasone response elements 1 and 2 (DexRE-1 and -2), respectively. Neither element has previously been associated with a glucocorticoid-activated transcriptional response. Conversion of the DexRE-1 to either a perfect thyroid hormone or vitamin D3 responsive element further enhanced induction by dexamethasone. Gel-shift analysis demonstrated that glucocorticoid receptor did not associate with either DexRE-1 or -2; hence, glucocorticoid receptor does not directly mediate glucocorticoid induction of CYP3A23. These unusual features suggest an alternate pathway through which glucocorticoids exert their effects.

Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24).

Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene.

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.

Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex.

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man.

Myc and Max: molecular evolution of a family of proto-oncogene products and their dimerization partner.

The myc gene family encodes a group of transcription factors that regulate cell proliferation and differentiation. These genes are widely studied because of their importance as proto-oncogenes. Phylogenetic analyses are described here for 45 Myc protein sequences representing c-, N-, L-, S-, and B-myc genes. A gene duplication early in vertebrate evolution produced the c-myc lineage and another lineage that later gave rise to the N- and L-myc lineages by another gene duplication. Evolutionary divergence in the myc gene family corresponds closely to the known branching order of the major vertebrate groups. The patterns of sequence evolution are described for five separate highly conserved regions, and these analyses show that differential rates of sequence divergence (= mosaic evolution) have occurred among conserved motifs. Further, the closely related dimerization partner protein Max exhibits significantly less sequence variability than Myc. It is suggested that the reduced variability in max stems from natural selection acting to preserve dimerization capability with products of myc and related genes.

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Analysis of perinatal gene expression: hormone response elements mediate activation of a lacZ reporter gene in liver of transgenic mice.

The transcription of genes encoding gluconeogenic enzymes is tightly regulated during the perinatal period. These genes are induced by glucagon (cAMP) and glucocorticoids and repressed by insulin. To address the role of cAMP and glucocorticoids in the physiological activation of genes encoding gluconeogenic enzymes in the perinatal period, transgenic mice have been generated with chimeric constructs containing the reporter gene lacZ under the control of hormone response elements. The activity of the transgene is restricted to the liver by the presence of the enhancers from the alpha-fetoprotein gene and its transcription is driven by a promoter that contains a TATA box linked to either cAMP response elements (CREs) or glucocorticoid response elements (GREs). We demonstrate cAMP and glucocorticoid regulation, liver-specific expression, and perinatal activation of the reporter gene. These data indicate that the CRE and GRE are, independently, necessary and sufficient to mediate perinatal gene activation. Perinatal activation was not impaired when a CRE reporter transgene was assayed in mice that contain a targeted mutation of the CRE-binding protein (CREB) gene, providing further evidence for functional redundancy among the members of the CREB/ATF gene family.

Plant terpenoid synthases: Molecular biology and phylogenetic analysis

This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library

Cancer/testis (CT) antigens—immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types—are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5′ end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Proviral insertions induce the expression of bone-specific isoforms of PEBP2αA (CBFA1): Evidence for a new myc collaborating oncogene

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2αA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2αA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2αA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2

Leishmania donovani is the etiologic agent of fatal visceral leishmaniasis in man. During their life cycle, Leishmania exist as flagellated promastigotes within the sandfly vector and as nonflagellated amastigotes in the macrophage phagolysosomal compartment of the mammalian host. The transformation from promastigotes to amastigotes is a critical step for the establishment of infection, and the molecular basis for this transformation is poorly understood. To define the molecular basis for amastigote survival in the mammalian host, we previously identified an amastigote stage-specific gene family termed “A2.” In the present study, we have inhibited the expression of A2 mRNA and A2 protein in amastigotes using antisense RNA and show that the resulting A2-deficient amastigotes are severely compromised with respect to virulence in mice. Amastigotes that did survive in the mice had restored A2 protein expression. These data demonstrate that A2 protein is required for L. donovani survival in a mammalian host, and this represents the first identified amastigote-specific virulence factor identified in Leishmania. This study also reveals that it is possible to study gene function in Leishmania through the expression of antisense RNA.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Protein–protein interactions among the Aux/IAA proteins

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic βαα DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the βαα containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis λ-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.