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PubMed2002

Bombyx mori orphan receptor, BmHR78: cDNA cloning, testis abundant expression and putative dimerization partner for Bombyx ultraspiracle.

We have identified a novel member of the nuclear receptor superfamily from the silkworm Bombyx mori, and named it as BmHR78, the B. mori hormone receptor. The DNA binding domain of BmHR78 shows high similarities to those of Tenebrio molitor hormone receptor 78, Drosophila hormone receptor 78, and mammalian testicular receptor 2, whereas the ligand binding domain is not well conserved. Northern blot analysis showed that BmHR78 gene was most abundantly expressed in the testis. From the fourth to fifth instar, BmHR78 gene was constantly expressed in the testis. In the anterior silk gland, the level of BmHR78 gene expression was developmentally changed. From day 10.0 to 11.0 in the fifth instar, another BmHR78 transcript with the smaller size appeared. Ultraspiracle (USP) isoform also appeared at the same stages in this tissue. BmHR78 forms not only a homodimer, but also a heterodimer with USP in a yeast two hybrid assay. The direct interaction between BmHR78 and USP was confirmed by pull down assay. Deletion mutant analysis showed that BmHR78 interacts with USP via the ninth heptad repeat in helix ten of the E region. This repeat is well conserved in RXR and its heterodimer partners, and shown to be an interface for their dimerization. In insect, only the ecdysone receptor and hormone receptor 38 are known thus far to dimerize with USP. Thus, BmHR78 is a third dimerization partner for USP and may modulate the molecular action of USP, including the ecdysone signal cascades.

Amino Acid Sequence↗
PubMed2006

AtGRXcp, an Arabidopsis chloroplastic glutaredoxin, is critical for protection against protein oxidative damage.

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage.

Amino Acid Sequence↗
PubMed1999

Analysis of the desmoplakin gene reveals striking conservation with other members of the plakin family of cytolinkers.

Members of the plakin family of cytolinker proteins integrate filaments into cellular networks and anchor these networks to the plasma membrane. Their importance is supported by the existence of cell and tissue fragility disorders caused by mutations in certain family members. In this study, the human gene encoding desmoplakin (DSP) was characterized and its structure compared with the related family members: plectin, bullous pemphigoid antigen 1 (BPAG1), envoplakin (EVPL) and periplakin (PPL). Sequence analysis of genomic clones was carried out in combination with a PCR-based strategy to define intron-exon borders. DSP was mapped using the GB4 radiation hybrid mapping panel to the interval between markers D6S296 and AFM043 x f2, corresponding to cytogenetic band 6p24. In addition, the murine gene (Dsp) was mapped to mouse chromosome 13 by interspecific backcross mapping. DSP encompasses approximately 45 kb organized into 24 exons and 23 introns, and the pattern of intron-exon borders bears a striking resemblance to other members of the plakin family. Notable features include the fact that a single large exon encodes the entire C-terminus of each gene. In contrast, the N-termini comprise numerous smaller exons with conservation of many intron-exon borders. Detailed characterization and mapping of these genes will facilitate their further evaluation as targets of genetic disorders and provide insights into the evolutionary relationships among molecules in this emerging gene family.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog.

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.

Alkyl and Aryl Transferases↗
PubMed2003

Y chromosome conserved anchored tagged sequences (YCATS) for the analysis of mammalian male-specific DNA.

Y chromosome haplotyping based on microsatellites or single nucleotide polymorphisms has recently proven to be a powerful approach for evolutionary studies of human populations, and also holds great promise for the studies of wild species. However, the use of the approach is hampered in most natural populations by the lack of Y chromosome markers and sequence information. Here, we report the large-scale development of Y chromosome conserved anchor tagged sequence (YCATS) markers in mammals by a polymerase chain reaction screening approach. Exonic primers flanking 48 different introns of Y-linked genes were developed based on human and mouse sequences, and screened on a set of 20 different mammals. On average about 10 introns were amplified for each species and a total of 100 kb of Y chromosome sequence were obtained. Intron size in humans was a reasonable predictor of intron size in other mammals (r2 = 0.45) and there was a negative correlation between human fragment size and amplification success. We discuss a number of factors affecting the possibility of developing conserved Y chromosome markers, including fast evolution of Y chromosome sequences due to male-biased mutation and adaptive evolution of male-specific genes, dynamic evolution of the Y chromosome due to being a nonrecombining unit, and homology with X chromosome sequences.

Animals↗
PubMed1998

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity.

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.

Alanine↗
PubMed2005

Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin.

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

Animals↗
PubMed2005

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification.

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.

Arginine↗
PubMed2002

Sequence requirements for terminators and antiterminators in the T box transcription antitermination system: disparity between conservation and functional requirements.

The T box transcription termination control system is used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase and other amino acid-related genes. Readthrough of a transcriptional terminator located in the leader region of the target gene is dependent on a specific interaction between the nascent leader transcript and the cognate uncharged tRNA. This interaction is required for formation of an antiterminator structure in the leader, which prevents formation of a competing transcriptional terminator stem-loop. The antiterminators and terminators of genes in this family are highly conserved in both secondary structure and primary sequence; the antiterminator contains the T box sequence, which is the most highly conserved leader element. These conserved features were investigated by phylogenetic and mutational analysis. Changes at highly conserved positions in the bulge and in the helix above the bulge reduced function, while alteration of other positions that were as much as 96% conserved did not have a major effect. The disparity between sequence conservation and function may be due to the requirement for maintaining base pairing in both the antiterminator and terminator structures.

Bacillus subtilis↗
PubMed1997

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Animals↗
PubMed1991

The evolution of rhodopsins and neurotransmitter receptors.

Rhodopsins share a limited number of amino acid identities with a variety of other integral membrane proteins. Most of these proteins have seven putative transmembrane segments and are likely to play a role in transmembrane signaling. We have undertaken a systematic series of comparisons of primary and secondary structure in order to clarify the functional and evolutionary significance of these sequence similarities. On the basis of consistently high similarity scores, we find that the most internally consistent definition of the rhodopsin gene family would include vertebrate rhodopsins, alpha- and beta-adrenergic receptors, M1 and M2 muscarinic acetylcholine receptors, substance K receptors, and insect rhodopsins, while excluding bacteriorhodopsin, the mass human oncogene, vertebrate and insect nicotinic acetylcholine receptors, and the yeast STE2 and STE3 peptide receptors. The rhodopsin gene family is highly diverged at the primary sequence level but has maintained a conserved secondary structure, including a previously unidentified hierarchy of transmembrane segment hydrophobicity. We have developed new computer algorithms for progressive multiple sequence alignment and the analysis of local conservation of protein domains, and we have used these algorithms to examine the phylogeny of the rhodopsin gene family and the changing domains of sequence conservation. The results show striking differences and similarities in the conserved domains in each of the three main branches of the rhodopsin gene family, and indicate that color vision arose independently in the lines of descent leading to modern humans and fruit flies.

Algorithms↗
PubMed2006

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis.

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.

Amino Acid Sequence↗
PubMed1986

Molecular model of the interaction between the glucocorticoid receptor and the regulatory elements of inducible genes.

Binding sites for the glucocorticoid receptor in an ecdysone-inducible gene from Drosophila melanogaster and in the chicken vitellogenin gene are described. A comparison with other binding sites for the glucocorticoid receptor, which have been analyzed by methylation protection experiments, shows they can be classified into three groups. The first group exhibits two blocks of contact points in two subsequent turns of the DNA helix, and includes only functional regulatory elements. The second group shows an identical contact with the hexanucleotide 5'-TGTYCT-3', but only half the contact points in the other turn of the helix, whereas the third group of sites exhibits only the contact points within the conserved hexanucleotide. An analysis of the hydrogen-bonding potential of the DNA base pairs along the major groove of 10 binding sites shows a very well-conserved pattern and a twofold rotational symmetry, suggesting that the array of hydrogen bonds may be a relevant aspect of sequence recognition by hormone receptors. A representation of the binding sites and contact points by computer graphics suggests the interaction of a receptor dimer, in a head-to-head arrangement, with two subsequent turns of the B-DNA helix within the glucocorticoid regulatory elements.

Animals↗
PubMed2003

A novel, divergent simian T-cell lymphotropic virus type 3 in a wild-caught red-capped mangabey (Cercocebus torquatus torquatus) from Nigeria.

We present here a novel, distinct simian T-cell lymphotropic virus (STLV) found in a red-capped mangabey (Cercocebus torquatus) (CTO-NG409), wild-caught in Nigeria, that showed an HTLV-2-like Western blot (WB) seroreactivity. The complete genome (8920 bp) of CTO-NG409 STLV was related to but different from STLV-3/PHA-PH969 (13.5 %) and STLV-3/PPA-F3 (7.6 %), and STLV-3/CTO604 (11.3 %), found in Eritrean and Senegalese baboons, and red-capped mangabeys from Cameroon, respectively. Phylogenetic analysis of a conserved tax (180 bp) sequence and the env gene (1482 bp) confirmed the relatedness of STLV-3/CTO-NG409 to the STLV-3 subgroup. Molecular clock analysis of env estimated that STLV-3/CTO-NG409 diverged from East and West/Central African STLV-3s about 140,900+/-12,400 years ago, suggesting an ancient African origin of STLV-3. Since phylogenetic evidence suggests multiple interspecies transmissions of STLV-1 to humans, and given the antiquity and wide distribution of STLV-3 in Africa, a search for STLV-3 in human African populations with HTLV-2-like WB patterns is warranted.

Animals↗
PubMed2005

Pattern similarity study of functional sites in protein sequences: lysozymes and cystatins.

BACKGROUND: Although it is generally agreed that topography is more conserved than sequences, proteins sharing the same fold can have different functions, while there are protein families with low sequence similarity. An alternative method for profile analysis of characteristic conserved positions of the motifs within the 3D structures may be needed for functional annotation of protein sequences. Using the approach of quantitative structure-activity relationships (QSAR), we have proposed a new algorithm for postulating functional mechanisms on the basis of pattern similarity and average of property values of side-chains in segments within sequences. This approach was used to search for functional sites of proteins belonging to the lysozyme and cystatin families. RESULTS: Hydrophobicity and beta-turn propensity of reference segments with 3-7 residues were used for the homology similarity search (HSS) for active sites. Hydrogen bonding was used as the side-chain property for searching the binding sites of lysozymes. The profiles of similarity constants and average values of these parameters as functions of their positions in the sequences could identify both active and substrate binding sites of the lysozyme of Streptomyces coelicolor, which has been reported as a new fold enzyme (Cellosyl). The same approach was successfully applied to cystatins, especially for postulating the mechanisms of amyloidosis of human cystatin C as well as human lysozyme. CONCLUSION: Pattern similarity and average index values of structure-related properties of side chains in short segments of three residues or longer were, for the first time, successfully applied for predicting functional sites in sequences. This new approach may be applicable to studying functional sites in un-annotated proteins, for which complete 3D structures are not yet available.

Amino Acid Sequence↗
PubMed2001

Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma-in-situ: analysis of European Organization for Research and Treatment of Cancer Trial 10853.

PURPOSE: In view of the increasing number of patients treated with breast-conserving treatment (BCT) for ductal carcinoma-in-situ (DCIS), risk factors for recurrence and metastasis should be identified. PATIENTS AND METHODS: Clinical and pathologic characteristics from patients with DCIS in the European Organization for Research and Treatment of Cancer trial 10853 (excision with or without radiotherapy) were related to the risk of recurrence. Pathologic features were derived from a central review of 863 of the 1,010 randomized cases (85%). The median follow-up was 5.4 years. RESULTS: Factors associated with an increased risk of local recurrence in the multivariate analysis were young age (< or = 40 years) (hazard ratio, 2.14; P =.02), symptomatic detection of DCIS (hazard ratio, 1.80; P =.008), growth pattern (solid and cribriform) (hazard ratios, 2.67 and 2.69, respectively; P =.012), involved margins (hazard ratio, 2.07; P =.0008), and treatment by local excision alone (hazard ratio, 1.74; P =.009). The risk of invasive recurrence was not related to the histologic type of DCIS (P =.63), but the risk of distant metastasis was significantly higher in poorly differentiated DCIS compared with well-differentiated DCIS (hazard ratio, 6.57; P =.01). CONCLUSION: Patients with poorly differentiated DCIS have a high risk of distant metastasis after invasive local recurrence. Margin status is the most important factor in the success of BCT for DCIS; additionally, young age and symptomatic detection of DCIS have negative prognostic value.

Adult↗
PubMed2001

Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity.

Despite extensive studies, there are still many unanswered questions regarding the mechanism of hedgehog signaling and the phylogenic conservation of hedgehog function in vertebrates. For example, whether hedgehog signaling in vertebrates requires smoothened is unclear, and the role of hedgehog activity in zebrafish is controversial. We show that inactivation of smoothened by retroviral insertions in zebrafish results in defects that are characteristic of hedgehog deficiencies, including abnormalities in body size, the central nervous system, adaxial mesoderm, cartilage and pectoral fins. We demonstrate that, as in Drosophila, vertebrate smoothened is essential for hedgehog signaling, and functions upstream of protein kinase A. Further analysis of neural tube defects revealed the absence of lateral floor plate and secondary motoneurons, but the presence of medial floor plate and primary motoneurons in smoothened mutant embryos. Blocking maternal hedgehog signaling by cyclopamine eliminates primary motoneurons, but not medial floor plate. Interestingly, even after inhibition of maternal hedgehog activity, the midbrain dopaminergic neurons still form, and looping of the heart does not randomize in the mutants. We also found decreased proliferation and increased apoptosis in the mutants. Taken together, these data demonstrate the conserved role of vertebrate smoothened in the hedgehog signaling pathway, and reveal similarities and differences of hedgehog function between teleosts and amniotes.

Animals↗
PubMed1996

Comparative and functional analysis of the AP2 promoter indicates that conserved octamer and initiator elements are critical for activity.

AP-2 is a developmentally-regulated transcription factor expressed in ectodermal cell lineages. The AP-2 protein is essential for neural tube, craniofacial and body wall morphogenesis and has been implicated in oncogenesis. Here we report the isolation of the AP-2 promoter from human, mouse and chicken. The initiation sites for the human gene have been mapped in a variety of cell lines, including several derived from breast tumours. Initiation occurs just upstream of an IR3-like repetitive element, present in the human and mouse genes, but absent in chicken. The cis-acting elements responsible for promoter activity in human HeLa cells have been mapped both in vivo and in vitro. The proximal promoter contains binding sites for transcription factors AP-2, NF-1 and octamer proteins, but lacks a TATA box motif. Functional analysis demonstrates that the octamer binding site is the critical component of basal promoter activity. In addition, the promoter relies on an initiator element for efficient start site utilization. There is an excellent correlation between the requirement for the initiator and octamer elements in transcription assays and the conservation of these cis-acting sequences between chicken, mouse and human.

Animals↗