C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.Single-stranded DNA (ssDNA)³-binding proteins have been assigned the role of removing secondary structure in DNA and protecting ssDNA from hydrolysis by nucleases (1). However, in addition to these mundane roles, ssDNA-binding proteins are now recognized as a key component of the replisome where they physically and functionally interact with other replication proteins and with the primer-template (2–4). ssDNA-binding proteins are also engaged in DNA recombination and repair (5). In view of these multiple roles, it has been difficult to identify the specific defect in genetically altered ssDNA-binding proteins that leads to an observed phenotype.The crystal structures of several prokaryotic ssDNA-binding proteins have been determined (6–8). These proteins have a conserved oligosaccharide-oligonucleotide binding fold (OB-fold) that is thought to bind the ssDNA by means of stacking and electrostatic interactions (6). Prokaryotic ssDNA-binding proteins also have an acidic C-terminal tail that is essential for bacterial and phage growth (9–13).The ssDNA-binding protein of bacteriophage T7 is encoded by gene 2.5 (14). The gene 2.5 protein (gp2.5) is a homodimer in solution, a structure that is stabilized by its C-terminal tail (9, 15). The C-terminal tail of one monomer of gp2.5 binds in a trans mode to the ssDNA-binding cleft of the other subunit, thus stabilizing the dimer interface observed in the crystal structure (6). The current model proposes that the positively charged DNA-binding cleft is shielded by the electrostatic charges of the C-terminal tail in the absence of ssDNA, thus facilitating oligomerization of gp2.5. Upon binding ssDNA, the dimer dissociates to allow the C-terminal tail to interact with other replication proteins (16). The tail modulates the affinity for ssDNA and protein-protein interactions by functioning as a two-way switch (6, 17). This mode of function is applicable to other prokaryotic ssDNA-binding proteins, namely Escherichia coli SSB protein and T4 gp32 (10, 13, 15, 18–22).gp2.5 is one of four proteins that include the T7 replisome. The other three proteins are the T7 gene 5 DNA polymerase (gp5), its processivity factor, E. coli thioredoxin (trx), and the multifunctional gene 4 helicase-primase (gp4). gp5 and trx bind with high affinity (K_D of 5 nm), and the two proteins are normally found in complex (gp5/trx) at a stoichiometry of one to one (23). The acidic C-terminal tail of gp2.5 is critical for the interactions of the protein with gp5/trx and gp4 (9, 24). The C-terminal tail binds to a positively charged segment located in the thumb subdomain of the gp5 (25). This fragment, designated the trx binding domain (TBD), is also the site of binding of the processivity factor, E. coli trx, and the C terminus of gp4. The multiple interactions of the C terminus of gp2.5 could thus function to coordinate the dynamic reactions occurring at the replication fork. gp2.5 is known to be critical for establishing coordination during leading and lagging strand DNA synthesis (26, 27).This C-terminal tail of gp2.5 is an acidic 26-amino acid segment with an aromatic phenylalanine as the C-terminal residue. The C-terminal tail is not seen in the crystal structure because gp2.5Δ26, lacking the tail, was used for crystallization; the wild-type protein did not yield crystals that diffracted (6). gp2.5ΔF designates a genetically modified gp2.5 lacking the C-terminal phenylalanine. gp2.5ΔF does not support the growth of T7Δ2.5 phage lacking gene 2.5 (28). Interestingly, T7 gene 4 protein also has an acidic C-terminal tail with a C-terminal phenylalanine (29). Again, the phenylalanine is critical for the interaction of gp4 with gp5/trx (29). Further evidence for overlapping binding sites of the C termini of these two proteins comes from studies with chimeric proteins (28, 29). The C-terminal tails of gp2.5 and gp4 can be exchanged, and the chimeric proteins support the growth of T7 phage lacking the corresponding wild-type protein.We recently designed a screen for suppressors of dominant lethal mutations of gp2.5 (30). The screen identified mutations in gene 5, the structural gene for T7 DNA polymerase (Fig. 1), which suppresses the lethal phenotype of gp2.5 mutant in which the C-terminal phenylalanine was moved to the penultimate position (gp2.5ΔF232InsF231). One of the altered suppressor genes (gp5, gp5-sup1) encodes a gp5 in which where glycine at position 371 is replaced by lysine (G371K). Whereas the other (gp5-sup2) encodes a protein in which threonine 258 and alanine 411 are replaced by methionine and threonine, respectively (T258M and A411T). The suppressor mutations in gp5 are necessary and sufficient to suppress the lethal phenotype of gp2.5ΔF232InsF231. The affected residues map in proximity to aromatic residues and to residues in close proximity to DNA as seen in the crystal structure of gp5/trx in complex with DNA (31). Throughout this study, gp2.5ΔF232InsF231 mutant will be referred to as gp2.5-FD because it effectively switches the positions of the C-terminal phenylalanine and the adjacent aspartic acid. E. coli SSB protein also has a C-terminal phenylalanine, and recent studies have shown that this residue inserts into a hydrophobic region consisting of exonuclease I of E. coli (45, 46).Open in a separate windowFIGURE 1.Amino acid changes in gp5 suppressor mutant polymerase(s). The amino acid changes in gp5 arising from the suppressor mutations in gene 5 are identified in the crystal structure of gp5/trx in complex with a primer-template and a nucleoside triphosphate (31). gp5 (light gray), trx (dark gray), and primer/template (red) are depicted. The suppressor mutation G371K (gp5-sup1) is shown in yellow and T258M and A411T (gp5-sup2) in orange.In this study, we have purified the two suppressor DNA polymerases and characterized them individually and in interaction with the other T7 replication proteins. Whereas wild-type gp5 binds with low affinity to gp2.5-FD, the DNA polymerases harboring the suppressor mutations bind with a higher affinity. An interesting finding is that whereas wild-type gp2.5 enables gp5/trx to catalyze strand displacement synthesis at a nick in DNA, gp2.5-FD does not support this reaction. Strand displacement synthesis is necessary for the initiation of leading strand DNA synthesis at a nick because it creates a 5′-single-stranded DNA tail for loading of the T7 helicase (32).     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Epstein-Barr Virus Lacking Glycoprotein gp42 Can Bind to B Cells but Is Not Able To Infect

The Epstein-Barr virus gH-gL complex includes a third glycoprotein, gp42, which is the product of the BZLF2 open reading frame (ORF). gp42 has been implicated as critical to infection of the B lymphocyte by virtue of its interaction with HLA class II on the B-cell surface. A neutralizing antibody that reacts with gp42 inhibits virus-cell fusion and blocks binding of gp42 to HLA class II; antibody to HLA class II can inhibit infection, and B cells that lack HLA class II can only be infected if HLA class II expression is restored. To confirm whether gp42 is an essential component of the virion, we derived a recombinant virus with a selectable marker inserted into the BZLF2 ORF to interrupt expression of the protein. A complex of gH and gL was expressed by the recombinant virus in the absence of gp42. Recombinant virus egressed from the cell normally and could bind to receptor-positive cells. It had, however, lost the ability to infect or transform B lymphocytes. Treatment with polyethylene glycol restored the infectivity of recombinant virus, confirming that gp42 is essential for penetration of the B-cell membrane.Entry of enveloped viruses into mammalian cells requires that the virion envelope fuse with the cell membrane after attachment to the cell surface. Herpesviruses require the functions of multiple protein species to mediate this event, and in keeping with the common origin and diverse habitats of these viruses, some of the proteins involved in penetration appear to be conserved throughout the family and some appear to be restricted to individual members or more closely related members with similar tropism. The two glycoproteins gH and gL fall into the first category of conserved proteins. Glycoprotein gH has been implicated as a major player in virus-cell fusion in many herpesviruses (8, 10, 11, 22, 28, 32, 34), and gL is an essential partner which is required for folding and transport of gH out of the endoplasmic reticulum (6, 19, 21, 27, 28, 35, 38, 45). The gH and gL homologs of Epstein-Barr virus (EBV) are gp85, the product of the BXLF2 open reading frame (ORF) (13, 31), and gp25, the product of the BKRF2 ORF (45), and these homologs appear to behave much as their counterparts in other herpesviruses do (45). However, a third glycoprotein, gp42, associates with the EBV gH-gL complex and falls into the second category of proteins, those with a more restricted distribution.Glycoprotein gp42 is the product of the BZLF2 ORF (26), and although there may be a functionally similar protein in cytomegalovirus (18, 24), it is not predicted to have a homolog in other human herpesviruses. It does, however, have a homolog in ORF51 of equine herpes virus 2 (43). Both EBV and equine herpes virus 2 infect B lymphocytes (1), and several lines of evidence suggest that, at least in the case of EBV, gp42 is critical to the infection of this cell type. A monoclonal antibody (MAb) called F-2-1 that reacts with gp42 has no affect on EBV attachment to its receptor, complement receptor type 2 (CR2) (CD21), but inhibits fusion of the virus with the B-cell membrane and neutralizes infection (29). Glycoprotein gp42 interacts with the β₁ domain of the HLA class II protein HLA-DR (39), and MAb F-2-1 interferes with this interaction (25). Like F-2-1, a MAb to HLA-DR or a soluble form of gp42 can block B-cell transformation, and B-cell lines which lack expression of HLA class II are not susceptible to superinfection with EBV unless expression of HLA class II is restored (25). Collectively these observations suggest that gp42, probably by virtue of its interaction with HLA class II, is essential to infection of the B lymphocyte. To answer directly the question of whether gp42 is an indispensable glycoprotein, we derived a virus that could be definitively shown to lack expression of the molecule and examined its ability to infect normal resting B lymphocytes. We report here that virus with expression of gp42 blocked can exit cells normally and can bind to receptor-positive target cells. However, it is unable to penetrate into cells and initiate infection.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]