The codon 47 polymorphism in p53 is functionally significant

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent serine 46 by the proline-directed kinase p38 MAPK is known to greatly enhance the ability of p53 to induce apoptosis. Here we showed that the serine 47 polymorphic variant, which replaces the proline residue necessary for recognition by proline-directed kinases, is a markedly poorer substrate for phosphorylation on serine 46 by p38 MAPK. Consistent with this finding, we showed that the serine 47 variant has up to 5-fold decreased ability to induce apoptosis compared with wild type p53. Mechanistically, we found that this variant has decreased ability to transactivate two p53 target genes, p53AIP1 and PUMA, but not other p53 response genes; this is the first time that phosphorylation of serine 46 has been implicated in transactivation of PUMA by p53. Down-regulation of PUMA in cells with wild type p53 using short interfering RNAs reduced apoptosis in these cells to a level comparable to that in cells containing the serine 47 variant. The combined data indicated that, like the codon 72 polymorphism, the codon 47 polymorphism of p53 is functionally significant and may play a role in cancer risk, progression, and the efficacy of therapy.     

The plasmodial surface anion channel is functionally conserved in divergent malaria parasites

The plasmodial surface anion channel (PSAC), a novel ion channel induced on human erythrocytes infected with Plasmodium falciparum, mediates increased permeability to nutrients and presumably supports intracellular parasite growth. Isotope flux studies indicate that other malaria parasites also increase the permeability of their host erythrocytes, but the precise mechanisms are unknown. Channels similar to PSAC or alternative mechanisms, such as the upregulation of endogenous host transporters, might fulfill parasite nutrient demands. Here we evaluated these possibilities with rhesus monkey erythrocytes infected with Plasmodium knowlesi, a parasite phylogenetically distant from P. falciparum. Tracer flux and osmotic fragility studies revealed dramatically increased permeabilities paralleling changes seen after P. falciparum infection. Patch-clamp of P. knowlesi-infected rhesus erythrocytes revealed an anion channel with striking similarities to PSAC: its conductance, voltage-dependent gating, pharmacology, selectivity, and copy number per infected cell were nearly identical. Our findings implicate a family of unusual anion channels highly conserved on erythrocytes infected with various malaria parasites. Together with PSAC's exposed location on the host cell surface and its central role in transport changes after infection, this conservation supports development of antimalarial drugs against the PSAC family.     

The H3 chaperone function of NASP is conserved in Arabidopsis

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1–H4 and H3.3–H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3–H4]₂ tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.     

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML–CysPc–C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc–C2L domains of land plant calpains form a separate sub‐clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1‐like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1‐3 mutant using CysPc–C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc–C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1‐3 mutant phenotype. In contrast, neither the CysPc–C2L domains from M. viride nor chimeric animal–plant calpains complement this mutant. Co‐evolution analysis identified differences in the interactions between the CysPc–C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1‐3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺‐binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.     

The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis

Ventral enclosure in Caenorhabditis elegans involves migration of epidermal cells over a neuroblast substrate and subsequent adhesion at the ventral midline. Organisation of the neuroblast layer by ephrins and their receptors is essential for this migration. We show that bus-8, which encodes a predicted glycosyltransferase, is essential for embryonic enclosure and acts in or with ephrin signalling to mediate neuroblast organisation and to permit epidermal migration. BUS-8 acts non-cell-autonomously in this process, and likely modifies an extracellular regulator of ephrin signalling and cell organisation. Weak and cold-sensitive alleles of bus-8 show that the gene has a separate and distinct post-embryonic role, being essential for epidermal integrity and production of the cuticle surface. This disorganisation of the epidermis and cuticle layers causes increased drug sensitivity, which could aid the growing use of C. elegans in drug screening and chemical genomics. The viable mutants are also resistant to infection by the pathogen Microbacterium nematophilum, due to failure of the bacterium to bind to the host surface. The two separate essential roles of BUS-8 in epidermal morphogenesis add to our growing understanding of the widespread importance of glycobiology in development.     

The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans

The Polycomb group (PcG) genes are required to maintain homeotic genes in a silenced state during development in drosophila and mammals and are thought to form several distinct silencing complexes that maintain homeotic gene repression during development. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. Although some PcG protein domains are conserved between flies and humans, substantial regions of several PcG proteins are divergent and humans contain multiple versions of each PcG gene. To determine the effects of these changes on the composition and function of a PcG complex, we have purified a human Polycomb repressive complex from HeLa cells (hPRC-H) that contains homologues of PcG proteins found in drosophila embryonic PRC1 (dPRC1). hPRC-H was found to have fewer components than dPRC1, retaining the PcG core proteins of dPRC1 but lacking most non-PcG proteins. Preparations of hPRC-H contained either two or three different homologues of most of the core PcG proteins, including a new Ph homologue we have named HPH3. Despite differences in composition, dPRC1 and hPRC-H have similar functions: hPRC-H is able to efficiently block remodeling of nucleosomal arrays through a mechanism that does not block the ability of nucleases to access and cleave the arrays.     

The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans

The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF(SM)) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AF(SM) depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.     

The splicing factor U2AF65 is functionally conserved in the thermotolerant deep-sea worm Alvinella pompejana

Due to their inherent stability, thermophilic bacteria and archaea serve as important resources for biochemical and biophysical analyses of many biological processes. Unfortunately, scientists characterizing eukaryote-specific processes, such as nuclear pre-mRNA splicing, are unable to take advantage of these sources of thermostable proteins. To identify and provide a source of thermostable eukaryotic proteins, we are characterizing splicing factors in the thermotolerant deep-sea vent polychaete, Alvinella pompejana. This worm, also known as the Pompeii worm, is found in the extreme environment of deep-sea hydrothermal vents, and is one of the most thermotolerant eukaryotic organisms known. We report on detailed analyses of U2AF65, the large subunit of the U2 small nuclear ribonucleoprotein auxiliary factor, an essential splicing factor important for intron definition and alternative splicing. The cloning and characterization of Pompeii U2AF65 show it is highly similar to human U2AF65 in sequence and function and is more thermostable than the human protein when bound to RNA in vitro. Notably, Pompeii U2AF65 can restore splicing in a human extract depleted of human U2AF. We also determine that the general splicing mechanisms and signal sequences are conserved in the Pompeii worm, an annelid which has previously been uncharacterized in terms of splicing factors and signals.     

The genomic structure of C14orf1 is conserved across eukarya

We have recently cloned the gene C14orf1, which is strongly expressed in normal testis and in several cancer cell lines and tumors. This gene maps to 14q24.3 and is interrupted by four introns. Two of them are also represented in the open reading frame of Schizosaccharomyces pombe in the same phase. In Arabidopsis taliana only the first of the two introns was found, in the same phase as the corresponding ones in S. pombe and human. Disruption of the ortholog in Saccharomyces cerevisiae (Yer044c) led to a severe growth defect, and C14orf1 failed to complement mutant yeast when put under the control of the natural Yer044c promoter. Further studies are needed to understand the causes underlying the high degree of conservation of the C14orf1 genomic structure. Received: 7 February 2000 / Accepted: 14 April 2000     

The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation

Single-celled cotton fiber (Gossypium hirsutum) provides a unique experimental system to study cell elongation. To investigate the role of the actin cytoskeleton during fiber development, 15 G. hirsutum ACTIN (GhACT) cDNA clones were characterized. RNA gel blot and real-time RT-PCR analysis revealed that GhACT genes are differentially expressed in different tissues and can be classified into four groups. One group, represented by GhACT1, is expressed predominantly in fiber cells and was studied in detail. A 0.8-kb GhACT1 promoter sufficient to confirm its fiber-specific expression was identified. RNA interference of GhACT1 caused significant reduction of its mRNA and protein levels and disrupted the actin cytoskeleton network in fibers. No defined actin network was observed in these fibers and, consequently, fiber elongation was inhibited. Our results suggested that GhACT1 plays an important role in fiber elongation but not fiber initiation.     

Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.     

Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein, hNPC2

Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the hNPC1 or hNPC2 gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2, Saccharomyces cerevisiae Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in hNPC2-/- patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.     

The novel pathogen-responsive glycosyltransferase UGT73C7 mediates the redirection of phenylpropanoid metabolism and promotes SNC1-dependent Arabidopsis immunity

Recent studies have shown that global metabolic reprogramming is a common event in plant innate immunity; however, the relevant molecular mechanisms remain largely unknown. Here, we identified a pathogen-induced glycosyltransferase, UGT73C7, that plays a critical role in Arabidopsis disease resistance through mediating redirection of the phenylpropanoid pathway. Loss of UGT73C7 function resulted in significantly decreased resistance to Pseudomonas syringae pv. tomato DC3000, whereas constitutive overexpression of UGT73C7 led to an enhanced defense response. UGT73C7-activated immunity was demonstrated to be dependent on the upregulated expression of SNC1, a Toll/interleukin 1 receptor-type NLR gene. Furthermore, in vitro and in vivo assays indicated that UGT73C7 could glycosylate p-coumaric acid and ferulic acid, the upstream metabolites in the phenylpropanoid pathway. Mutations that lead to the loss of UGT73C7 enzyme activities resulted in the failure to induce SNC1 expression. Moreover, glycosylation activity of UGT73C7 resulted in the redirection of phenylpropanoid metabolic flux to biosynthesis of hydroxycinnamic acids and coumarins. The disruption of the phenylpropanoid pathway suppressed UGT73C7-promoted SNC1 expression and the immune response. This study not only identified UGT73C7 as an important regulator that adjusts phenylpropanoid metabolism upon pathogen challenge, but also provided a link between phenylpropanoid metabolism and an NLR gene.     

The box C/D sRNP dimeric architecture is conserved across domain Archaea

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.