IL-22 is an IL-10 homologue that binds to and signals through the class II cytokine receptor heterodimer IL-22RA1/CRF2-4. IL-22 is produced by T cells and induces the production of acute-phase ...reactants in vitro and in vivo, suggesting its involvement in inflammation. Here we report the identification of a class II cytokine receptor designated IL-22RA2 (IL-22 receptor-α 2) that appears to be a naturally expressed soluble receptor. IL-22RA2 shares amino acid sequence homology with IL-22RA1 (also known as IL-22R, zcytor11, and CRF2-9) and is physically adjacent to IL-20Rα and IFN-γR1 on chromosome 6q23.3-24.2. We demonstrate that IL-22RA2 binds specifically to IL-22 and neutralizes IL-22-induced proliferation of BaF3 cells expressing IL-22 receptor subunits. IL-22RA2 mRNA is highly expressed in placenta and spleen by Northern blotting. PCR analysis using RNA from various tissues and cell lines showed that IL-22RA2 was expressed in a range of tissues, including those in the digestive, female reproductive, and immune systems. In situ hybridization revealed the dominant cell types expressing IL-22RA2 were mononuclear cells and epithelium. Because IL-22 induces the expression of acute phase reactants, IL-22RA2 may play an important role as an IL-22 antagonist in the regulation of inflammatory responses.
The uptake of iron in plants is a highly regulated process that is induced on iron starvation. In tomato, the mutant chloronerva exhibits constitutive expression of iron uptake responses and ...intercostal chlorosis. Biochemically, chloronerva is an auxotroph for nicotianamine, a key polyamine in plant iron uptake metabolism. The chloronerva gene has been fine-mapped onto the long arm of chromosome 1 in a large segregating tomato population and yeast artificial chromosome clones encompassing the region were isolated by using flanking markers. A cosmid contig containing the chloronerva gene was established, and complementing cosmids were identified by transformation into the mutant. The chloronerva transcript was identified by cDNA isolation using the complementing cosmids. The gene encodes a unique protein of 35 kDa. The mutant harbors a single base change compared with the wild type. Based on enzyme activity and sequence similarity to the coding DNA sequence of the purified barley enzyme the chloronerva gene encodes the enzyme nicotianamine synthase.
The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ...ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3′flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5′upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.
The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal ...expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10-10 M dihydrotestosterone or 10-9 M testosterone, but not 10-6 M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.
Friedreich's ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence ...corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for ≈ 17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through ``premutation'' intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.
The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a λ gt11 cDNA expression library with a 9ORF1 protein probe yielded a ...novel cellular PDZ domain-containing protein, 9BP-1, which binds to wildtype, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo, with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.
A 2,479-base pair mitochondrial DNA fragment was sequenced for eight chromosome races of Sceloporus grammicus from central Mexico to estimate their phylogenetic relationships. The species S. ...poinsetti and S. olivaceus were used separately as alternative outgroups. A total of 795 positions varied in three complete protein-coding genes examined (ND3, ND4L, ND4), and 52 of 292 positions varied across five transfer RNAs examined (glycine, argenine, histidine, serine, leucine). Sequence divergence values ranged from 0.0 to 0.23 among the ingroup taxa, and a maximum of 0.26 was observed between ingroup and outgroup taxa. Alternative analyses based upon equally weighted characters and several alternative character-weighting options were used to obtain phylogenetic hypotheses for the complex, and a single most-parsimonious tree was selected from among these on the basis of a new character-weighting method that takes into account the observed frequencies of all 12 possible substitutions for protein genes. The most-parsimonious cladogram showed that chromosomal evolution in this complex has been more complicated than previously hypothesized. Several rearrangements (Robertsonian fissions) have evolved independently on two or more occasions, which corroborates evidence from other studies showing that single rearrangements are not underdominant upon their origin, and their fixation probabilities are enhanced by repeated origins. These observations refute expectations of some general models of chromosome evolution. The same phylogenetic hypothesis was used to test the minimum-interaction model of chromosome evolution and a specific model for the evolution of macrochromosome 2. A clear distinction was also possible among alternative hypotheses of relationship for three chromosome races involved in hybridization, and the consequences for the role of chromosomal rearrangements in reducing gene flow are discussed in this context.
The article analyzes the evolution of the Amsterdam capital market as a consequence of Dutch overseas expansion and the introduction of transferable VOC shares. Offering investors prospects of ...speculative gains without serious loss of liquidity, these instruments created a booming secondary market offering a wide range of allied credit techniques. By 1609 this market had become sufficiently strong to dictate terms for new public debt issues. These findings show that, contrary to commonly held notions about the emergence of secondary markets, private finance took precedence over public finance in the Dutch Republic.
Intron-containing pre-mRNAs are normally retained in the nucleus until they are spliced to produce mature mRNAs that are exported to the cytoplasm. Although the detailed mechanism is not well ...understood, the formation of splicing-related complexes on pre-mRNAs is thought to be responsible for the nuclear retention. Therefore, pre-mRNAs containing suboptimal splice sites should tend to leak out to the cytoplasm. Such pre-mRNAs often contain purine-rich exonic splicing enhancers (ESEs) that stimulate splicing of the adjacent intron. Here, we show that ESEs per se possess an activity to retain RNAs in the nucleus through a saturable nuclear retention factor. Cross-competition experiments revealed that intron-containing pre-mRNAs (without ESEs) used the same saturable nuclear retention factor as ESEs. Interestingly, although intronless mRNAs containing ESEs were also poorly exported, spliced mRNAs produced from ESE-containing pre-mRNAs were efficiently exported to the cytoplasm. Thus, the splicing reaction can reset the nuclear retention state caused by ESEs, allowing nuclear export of mature mRNAs. Our results reveal a novel aspect of ESE activity that should contribute to gene expression and RNA quality control.
A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major ...β/A4-protein (Aβ) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-Aβ component of AD amyloid) and its precursor NACP. NAC is the second component, after Aβ, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form β-structures consistent with its association with amyloid. NACP is detected as a Mr19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.
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