Genes linked by fusion events are generally of the same functional category: A systematic analysis of 30 microbial genomes

Recent work in computational genomics has shown that a functional association between two genes can be derived from the existence of a fusion of the two as one continuous sequence in another genome. For each of 30 completely sequenced microbial genomes, we established all such fusion links among its genes and determined the distribution of links within and among 15 broad functional categories. We found that 72% of all fusion links related genes of the same functional category. A comparison of the distribution of links to simulations on the basis of a random model further confirmed the significance of intracategory fusion links. Where a gene of annotated function is linked to an unclassified gene, the fusion link suggests that the two genes belong to the same functional category. The predictions based on fusion links are shown here for Methanobacterium thermoautotrophicum, and another 661 predictions are available at http://fusion.bu.edu.

Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state.

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.

Observation via one-dimensional 13Calpha NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper.

Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Calpha at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (> >2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is > or= 10-20 ms.

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n of fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor.

The estrogen receptor (ER), a 66-kDa protein that mediates the actions of estrogens in estrogen-responsive tissues, is a member of a large superfamily of nuclear hormone receptors that function as ligand-activated transcription factors. ER shares a conserved structural and functional organization with other members of this superfamily, including two transcriptional activation functions (AFs), one located in its amino-terminal region (AF-1) and the second located in its carboxyl-terminal, ligand-binding region (AF-2). In most promoter contexts, synergism between AF-1 and AF-2 is required for full ER activity. In these studies, we demonstrate a functional interaction of the two AF-containing regions of ER, when expressed as separate polypeptides in mammalian cells, in response to 17 beta-estradiol (E2) and antiestrogen binding. The interaction was transcriptionally productive only in response to E2, and was eliminated by point or deletion mutations that destroy AF-1 or AF-2 activity or E2 binding. Our results suggest a definitive mechanistic role for E2 in the activity of ER--namely, to alter receptor conformation to promote an association of the amino- and carboxyl-terminal regions, leading to transcriptional synergism between AF-1 and AF-2. The productive re assembly of two portions of ER expressed in cells as separate polypeptides demonstrates the evolutionarily conserved modular structural and functional organization of the nuclear hormone receptors. The ligand-dependent interaction of the two AF-containing regions of ER allows for the assembly of a complete activation function from two distinct regions within the same protein, providing a mechanism for hormonally regulated transcription.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Multiple origins of the yucca-yucca moth association.

The association of species of yucca and their pollinating moths is considered one of the two classic cases of obligate mutualism between floral hosts and their pollinators. The system involves the active collection of pollen by females of two prodoxid moth genera and the subsequent purposeful placement of the pollen on conspecific stigmas of species of Yucca. Yuccas essentially depend on the moths for pollination and the moths require Yucca ovaries for oviposition. Because of the specificity involved, it has been assumed that the association arose once, although it has been suggested that within the prodoxid moths as a whole, pollinators have arisen from seed predators more than once. We show, by using phylogenies generated from three molecular data sets, that the supposed restriction of the yucca moths and their allies to the Agavaceae is an artifact caused by an incorrect circumscription of this family. In addition we provide evidence that Yucca is not monophyletic, leading to the conclusion that the modern Yucca-yucca moth relationship developed independently more than once by colonization of a new host.

The pre-S domain of the large viral envelope protein determines host range in avian hepatitis B viruses.

In addition to their well-recognized hepatotropism, all hepatitis B viruses (HBVs) display marked species specificity, growing poorly or not at all in species other than those closely related to their natural hosts. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. HHBV virions will not infect ducks in vivo and infect cultured duck hepatocytes extremely inefficiently in vitro. Mutant HHBV genomes lacking all viral envelope proteins (HHBV env-) can be complemented in trans with DHBV envelope proteins; the resulting pseudotyped virions can efficiently infect duck hepatocytes. Further complementation analysis reveals that of the two viral surface proteins (L and S), it is the L protein that determines host range. Pseudotyping of HHBV env- with DHBV/HHBV chimeric envelope proteins reveals that replacement of as few as 69 amino acids of the pre-S domain of the HHBV L protein by their DHBV counterparts is sufficient to permit infection of duck hepatocytes. These studies indicate that the species-specificity of hepadnaviral infection is determined at the level of virus entry and is governed by the pre-S domain of the viral L protein.

Location of light-repressible, small GTP-binding protein of the YPT/rab family in the growing zone of etiolated pea stems.

YPT/rab proteins are ras-like small GTP-binding proteins that serve as key regulators of vesicular transport. The mRNA levels of two YPT/rab genes in pea plants are repressed by light, with the process mediated by phytochrome. Here, we examined the mRNA expression and the location of the two proteins, pra2- and pra3-encoded proteins, using monoclonal antibodies. The pra2 and pra3 mRNA levels were highest in the stems of dark-grown seedlings. The corresponding proteins were found in the cytosol and the membranes of the stems. Most of the pra2 protein was in the growing internodes, especially in the growing region, but the pra3 protein was widespread. These results suggest that the pra2 protein is important for vesicular transport in stems, possibly contributing to stem growth in the dark, and that the pra3 protein is important for general vesicular transport. The amounts of pra2 and pra3 proteins decreased with illumination. The decrease in these proteins may be related to the phytochrome-dependent inhibition of stem growth that occurs in etiolated pea seedlings.

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.

Swapping structural determinants of ribonucleases: an energetic analysis of the hinge peptide 16-22.

Bovine seminal ribonuclease (BS-RNase) is a homodimeric enzyme strictly homologous to the pancreatic ribonuclease (RNase A). Native BS-RNase is an equilibrium mixture of two distinct dimers differing in the interchange of the N-terminal segments and in their biological properties. The loop 16-22 plays a fundamental role on the relative stability of the two isomers. Both the primary and tertiary structures of the RNase A differ substantially from those of the seminal ribonuclease in the loop region 16-22. To analyze the possible stable conformations of this loop in both enzymes, structure predictions have been attempted, according to a procedure described by Palmer and Scheraga [Palmer, K. A. & Scheraga, H. A. (1992) J. Comput. Chem. 13, 329-350]. Results compare well with experimental x-ray structures and clarify the structural determinants that are responsible for the swapping of the N-terminal domains and for the peculiar properties of BS-RNase. Minimal modifications of RNase A sequence needed to form a stable swapped dimer are also predicted.