Amino Acid Substitution Mutants Research Articles

Compound Heterozygous Desmoplakin Mutations Result in a Phenotype with a Combination of Myocardial, Skin, Hair, and Enamel Abnormalities

Desmoplakin (DP) anchors the intermediate filament cytoskeleton to the desmosomal cadherins and thereby confers structural stability to tissues. In this study, we present a patient with extensive mucocutaneous blisters, epidermolytic palmoplantar keratoderma, nail dystrophy, enamel dysplasia, and sparse woolly hair. The patient died at the age of 14 years from undiagnosed cardiomyopathy. The skin showed hyperplasia and acantholysis in the mid- and lower epidermal layers, whereas the heart showed extensive fibrosis and fibrofatty replacement in both ventricles. Immunofluorescence microscopy showed a reduction in the C-terminal domain of DP in the skin and oral mucosa. Sequencing of the DP gene showed undescribed mutations in the maternal and paternal alleles. Both mutations affected exon 24 encoding the C-terminal domain. The paternal mutation, c.6310delA, leads to a premature stop codon. The maternal mutation, c.7964 C to A, results in a substitution of an aspartic acid for a conserved alanine residue at amino acid 2655 (A2655D). Structural modeling indicated that this mutation changes the electrostatic potential of the mutated region of DP, possibly altering functions that depend on intermolecular interactions. To conclude, we describe a combination of DP mutation phenotypes affecting the skin, heart, hair, and teeth. This patient case emphasizes the importance of heart examination of patients with desmosomal genodermatoses.

Serine-Threonine Kinase Receptor-associated Protein Inhibits Apoptosis Signal-regulating Kinase 1 Function through Direct Interaction

Serine-threonine kinase receptor-associated protein (STRAP) interacts with transforming growth factor beta (TGF-beta) receptors and inhibits TGF-beta signaling. Here, we identify STRAP as an interacting partner of ASK1 (apoptosis signal-regulating kinase 1). The association between ASK1 and STRAP is mediated through the C-terminal domain of ASK1 and the fourth and sixth WD40 repeats of STRAP. Using cysteine-to-serine amino acid substitution mutants of ASK1 (C1005S, C1351S, C1360S, and C1351S/C1360S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that Cys(1351) and Cys(1360) of ASK1 and Cys(152) and Cys(270) of STRAP are required for ASK1-STRAP binding. ASK1 phosphorylated STRAP at Thr(175) and Ser(179), suggesting a potential role for STRAP phosphorylation in ASK1 activity regulation. Expression of wild-type STRAP, but not STRAP mutants (C152S/C270S and T175A/S179A), inhibited ASK1-mediated signaling to both JNK and p38 kinases by stabilizing complex formation between ASK1 and its negative regulators, thioredoxin and 14-3-3, or decreasing complex formation between ASK1 and its substrate MKK3. In addition, STRAP suppressed H(2)O(2)-mediated apoptosis in a dose-dependent manner by inhibiting ASK1 activity through direct interaction. These results suggest that STRAP can act as a negative regulator of ASK1.

Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production

The adenovirus type 5 (Ad5) late region 4 (L4) 100-kDa nonstructural protein (L4-100K) mediates inhibition of cellular protein synthesis and selective translation of tripartite leader (TL)-containing viral late mRNAs via ribosome shunting. In addition, L4-100K has been implicated in the trimerization and nuclear localization of hexon protein. We previously proved that L4-100K is a substrate of the protein arginine methylation machinery, an emergent posttranslational modification system involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing, and DNA repair. As understood at present, L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. To identify the methylated arginine residues and assess the role of L4-100K arginine methylation, we generated amino acid substitution mutations in the RGG and GAR motifs to examine their effects in Ad-infected and plasmid-transfected cells. Arginine-to-glycine exchanges in the RGG boxes significantly diminished L4-100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation in RGG motifs is an important host cell function required for efficient Ad replication. Our data further indicate that PRMT1-catalyzed arginine methylation in the RGG boxes regulates the binding of L4-100K to hexon and promotes the capsid assembly of the structural protein as well as modulating TL-mRNA interaction. Furthermore, substitutions in GAR, but not RGG, regions affected L4-100K nuclear import, implying that the nuclear localization signal of L4-100K is located within the GAR sequence.

Are Affinity-Enhanced T Cells the Future of HIV Therapy?

, suggesting that under rare circumstances HIV-1 infection can be effectively controlled by a protective T-cell response.T-cell exhaustion has been most thoroughly studied using the lymphocytic choriomenin-gitis virus (LCMV) model. Mice clear wild-type LCMV infection rapidly and protective memory develops. By contrast, a two amino-acid substitu-tion mutant of LCMV (Clone 13) is not cleared and a chronic, persistent infection ensues

Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881C>A (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined.

MDia2 Shuttles between the Nucleus and the Cytoplasm through the Importin-α/β- and CRM1-mediated Nuclear Transport Mechanism

Mammalian homolog of Drosophila diaphanous (mDia) consisting of three isoforms, mDia1, mDia2, and mDia3, is an effector of Rho GTPases that catalyzes actin nucleation and polymerization. Although the mDia actions on actin dynamics in the cytoplasm have been well studied, whether mDia accumulates and functions in the nucleus remains largely unknown. Given the presence of actin and actin-associated proteins in the nucleus, we have examined nuclear localization of mDia isoforms. We expressed each of mDia isoforms as a green fluorescent protein fusion protein and examined their localization. Although all the mDia isoforms were localized predominantly in the cytoplasm under the steady-state conditions, mDia2 and not mDia1 or mDia3 accumulated extensively in the nucleus upon treatment with leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The LMB-induced nuclear accumulation was confirmed for endogenous mDia2 by using an antibody specific to mDia2. Studies using green fluorescent protein fusions of various truncation mDia2 mutants and point mutants of some of these proteins identified a functional nuclear localization signal in the N terminus of mDia2 and at least one functional nuclear export signal in the C terminus. The nuclear localization signal of mDia2 bound to importin-alpha and was imported into the nucleus by importin-alpha/beta complex in an in vitro transport assay. Consistently, depletion of importin-beta with RNA interference suppressed the LMB-induced nuclear localization of endogenous mDia2. These results suggest that mDia2 continuously shuttles between the nucleus and the cytoplasm using specific nuclear transport machinery composing of importin-alpha/beta and CRM1.

A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti

To identify pertinent mutations associated with knockdown resistance to permethrin, the entire coding sequence of the voltage-gated sodium channel gene Aa-para was sequenced and analyzed from a Per-R strain with 190-fold resistance to permethrin and two susceptible strains of Aedes aegypti. The longest transcript, a 6441bp open reading frame, encodes 2147 amino acid residues with an estimated molecular mass of 241kDa. A total of 33 exons were found in the Aa-para gene over 293kb of genomic DNA. Three previously unreported optional exons were identified. The first two exons, m and n, were located within the intracellular domain I/II, and the third, f′, was found within the II/III linkers. The two mutually exclusive exons, d and l, were the only alternative exons in all the cDNA clones sequenced in this study. The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain. The high frequency and coexistence of the two mutations in the Per-R strain suggest that they might exert a synergistic effect to provide the knockdown resistance to permethrin. Furthermore, both cDNA and genomic DNA data from the same individual mosquitoes have demonstrated that RNA editing was not involved in amino acid substitutions of the Per-R strain.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro

RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.

Physiological and Biochemical Defects in Carboxyl-terminal Mutants of Mitochondrial DNA Helicase

Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.

HBP21: A Novel Member of TPR Motif Family, as a Potential Chaperone of Heat Shock Protein 70 in Proliferative Vitreoretinopathy (PVR) and Breast Cancer

A large number of tetratricopeptide repeat (TPR)-containing proteins have been shown to interact with the C-terminal domain of the 70 kDa heat-shock protein (Hsp70), especially those with three consecutive TPR motifs. The TPR motifs in these proteins are necessary and sufficient for mediating the interaction with Hsp70. Here, we investigate HBP21, a novel human protein of unknown function having three tandem TPR motifs predicted by computational sequence analysis. We confirmed the high expression of HBP21 in breast cancer and proliferative vitreoretinopathy (PVR) proliferative membrane and examined whether HBP21 could interact with Hsp70 using a yeast two-hybrid system and glutathione S-transferase pull-down assay. Previous studies have demonstrated the importance of Hsp70 C-terminal residues EEVD and PTIEEVD for interaction with TPR-containing proteins. Here, we tested an assortment of truncation and amino acid substitution mutants of Hsp70 to determine their ability to bind to HBP21 using a yeast two-hybrid system. The newly discovered interaction between HBP21 and Hsp70 along with observations from other studies leads to our hypothesis that HBP21 may be involved in the inhibition of progression and metastasis of tumor cells.

Heat Shock Protein 90 Inhibition in Lung Cancer

The heat shock protein 90 (Hsp90) chaperone is required for the conformational maturation and stability of multiple oncogenic kinases that drive signal transduction and proliferation of lung cancer cells. The recent demonstration that mutant epidermal growth factor receptor is an Hsp90 client, irrespective of the presence of the secondary threonine-to-methionine amino acid substitution mutation at position 790 mediating anilinoquinazoline resistance, suggests Hsp90 inhibition as a novel strategy against this group of lung cancers. The rarer epidermal growth factor receptors harboring exon 20 insertions and vIII mutations are also Hsp90 clients. Lung cancers may also be driven by mutant ErbB2, mutant B-Raf, or mutant or overexpressed c-Met, all of which are also degraded on Hsp90 inhibition. Hsp90 inhibitors may be synergistic with other drugs that disrupt chaperone function, including inhibitors of histone deacetylase 6 and the proteasome and agents that inhibit Hsp70 function. Hsp90 plays a unique antiapoptotic role in small cell lung cancer cells, so that Hsp90 inhibition results in substantial cell death in both chemosensitive and chemoresistant small cell lung cancer cell lines. Clinically, the geldanamycin compounds are the most mature, with manageable toxic effects. Several new classes of Hsp90 inhibitors are emerging, including purines and pyrazoles that have entered phase 1 trials. The available data suggest that Hsp90 inhibitors should be evaluated in multiple lung cancer subsets.

Identification of Alix-type and Non-Alix-type ALG-2-binding Sites in Human Phospholipid Scramblase 3: DIFFERENTIAL BINDING TO AN ALTERNATIVELY SPLICED ISOFORM AND AMINO ACID-SUBSTITUTED MUTANTS

ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.

Site‐directed mutagenesis of bovine deoxyhypusine hydroxylase at the hinge loop area

Deoxyhypusine hydroxylase (DOHH) is the second of the two enzymes that catalyze the maturation of eukaryotic initiation factor 5A (eIF5A). It has been well established that hypusinated eIF5A is essential for eukaryotic cell proliferation and survival and that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy.We have recently cloned bovine DOHH cDNA. The deduced amino acid sequence shared 88% identity with human DOHH. The dual function of human DOHH has been reported: the Fe containing holoenzyme exhibited hydroxylase activity while the apoenzyme showed molecular chaperone activity (Kang et al., The FASEB Journal. 2007;21:lb122). The structure transition between the holoenzyme and the apoenzyme is believed to take place around the variable hinge loop area. In this work, we prepare three amino acid substitution mutants at the loop area: P143S (Pro at the position 143 to Ser), D148A (Asp at 148 to Ala), and E154V (Glu at 154 to Val) of the bovine DOHH. The mutant DNA has been created by site‐directed mutagenesis using PCR primed with mutagenic primers followed by overlap extension. The mutant DNA is subcloned into pGEX‐2T, overexpressed, and characterized. We will compare the hydroxylase and chaperone activities of the mutant proteins to the wild‐type DOHH.Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.

Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin

Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity.

Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.

A bridge to transcription by RNA polymerase

A comprehensive survey of single amino-acid substitution mutations critical for RNA polymerase function published in Journal of Biology supports a proposed mechanism for polymerase action in which movement of the polymerase 'bridge helix' promotes transcriptional activity in cooperation with a critical substrate-interaction domain, the 'trigger loop'.

Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P(2) depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P(2) depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P(2)-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P(2). To examine a putative Gag interaction with PI(4,5)P(2), we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P(2). Using this assay, we observed that PI(4,5)P(2) significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P(2) for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P(2) binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P(2)-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P(2) on the membrane and that the MA basic domain mediates this interaction.

Drosophila follicle cell amplicons as models for metazoan DNA replication: A cyclinE mutant exhibits increased replication fork elongation

Gene clusters amplified in the ovarian follicle cells of Drosophila serve as powerful models for metazoan DNA replication. In response to developmental signals, specific genomic regions undergo amplification by repeated firing of replication origins and bidirectional movement of replication forks for approximately 50 kb in each direction. Previous work focused on initiation of amplification, defining replication origins, establishing the role of the prereplication complex and origin recognition complex (ORC), and uncovering regulatory functions for the Myb, E2F1, and Rb transcription factors. Here, we exploit follicle cell amplification to investigate the control of DNA replication fork progression and termination, poorly understood processes in metazoans. We identified a mutant in which, during gene amplification, the replication forks move twice as far from the origin compared with wild type. This phenotype is the result of an amino acid substitution mutation in the cyclinE gene, cyclinE(1f36). The rate of oogenesis is normal in cyclinE(1f36)/cyclinE(Pz8) mutant ovaries, indicating that increased replication fork progression is due to increased replication fork speed, possibly from increased processivity. The increased amplification domains observed in the mutant imply that there are not replication fork barriers preventing replication forks from progressing beyond the normal 100-kb amplified region. These results reveal a previously unrecognized role for CyclinE in controlling replication fork movement.

Genetic and Phenotypic Identification of Fusidic Acid-Resistant Mutants with the Small-Colony-Variant Phenotype in Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus are a slow-growing subpopulation whose phenotypes can include resistance to aminoglycosides, defects in electron transport, and enhanced persistence in mammalian cells. Here we show that a subset of mutants selected as SCVs by reduced susceptibility to aminoglycosides are resistant to the antibiotic fusidic acid (FA) and conversely that a subset of mutants selected for resistance to FA are SCVs. Mutation analysis reveals different genetic classes of FA-resistant SCVs. One class, FusA-SCVs, have amino acid substitution mutations in the ribosomal translocase EF-G different from those found in classic FusA mutants. Most of these mutations are located in structural domain V of EF-G, but some are in domain I or III. FusA-SCVs are auxotrophic for hemin. A second class of FA-resistant SCVs carry mutations in rplF, coding for ribosomal protein L6, and are designated as FusE mutants. FusE mutants fall into two phenotypic groups: one auxotrophic for hemin and the other auxotrophic for menadione. Accordingly, we have identified new genetic and phenotypic classes of FA-resistant mutants and clarified the genetic basis of a subset of S. aureus SCV mutants. A clinical implication of these data is that FA resistance could be selected by antimicrobial agents other than FA.