Showing papers in "Genes to Cells in 1996"

Phosphorylation of Ser‐3 of cofilin regulates its essential function on actin

Abstract: Background: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. Results: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F-or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient overexpression of wild-type or S3A-cofilin in cultured cells caused disruption of pre-existing actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the overexpression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with Δcof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. Conclusion: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the functions of unphosphorylated cofilin for viability.

Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway.

Abstract: Background: Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of molecular chaperones and folding enzymes localized in the ER. Thus, eukaryotic cells possess an intracellular signalling pathway from the ER to the nucleus, called the unfolded protein-response (UPR) pathway. In Saccharomyces cerevisiae, such induction is mediated by the cis-acting unfolded protein-response element (UPRE) which has been thought to be recognized by one or more transcription factor(s). Results: Extensive mutational analysis revealed that UPRE contains a partial palindrome with a spacer of one nucleotide (CAGCGTG) that is essential for its function. We then cloned the ERN4 (presumably identical with HAC1) gene using yeast one-hybrid screening, in which the GAL4-ERN4 fusion gene constitutively activates the UPR pathway. The ERN4 gene encodes a basic-leucine zipper protein (Ern4p) that specifically binds to UPRE in vitro and activates transcription in vivo. Cells lacking Ern4p are unable to induce transcription of any of the five target genes tested and exhibit sensitivity to ER stress and inositol requirement for growth. Conclusion: We concluded that Ern4p represents a major component of the putative transcription factor (UPRF) responsible for the UPR leading to the induction of ER-localized stress proteins.

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

Abstract: Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA. Starting with genomic negative sense (−) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low. Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (−)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (−)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5′ ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein. Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (−)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.

Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1

Abstract: Background: Ad4BP/SF-1 was originally identified as a steroidogenic tissue-specific transcription factor. Recent gene disruption studies with the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 clearly revealed the essential function of the factor for adrenal and gonadal differentiation. Results: In this study, we examined the early development of these tissues using Ad4BP/SF-1 as the marker. In rat foetuses of 11.5 days post-coitum (d.p.c.), a cell population designated adreno-genital primordium was firstly observed on symmetrical lines extending from the dorsal aorta to the dorsal coelomic epithelia of the primitive urogenital ridges. From 12.5 d.p.c., the rostral half of the adreno-genital primordium started to separate into two distinct cell populations. Judging from the distribution of primordial germ cells, the cell population on the dorsal aortal side is a primordium for the adrenal cortex whereas that on the coelomic epithelial side is for the gonads. At 13.5 d.p.c., these two primordia have separated completely. Conclusion: These observations clearly identified a novel adreno-genital primordium from which both the adrenal cortex and the gonads originate. An RT-PCR study conducted to detect adrenal- and gonad-specific mRNAs supported the above observations.

A yeast gene product, Fob1 protein, required for both replication fork blocking and recombinational hotspot activities

Abstract: Background: In the rRNA gene cluster on the Saccharomyces cerevisiae chromosome XII, a unique site that blocks progression of the replication fork exists in a single unit of the repeats. The site RFB (replication fork blocking) is located within one of two cis-elements present in a nontranscribed region of each repeated unit, which are required for obtaining maximal activity of a hotspot (HOT1) dependent recombination. Results: To investigate the correlation between replication fork blocking at RFB and homologous recombination at HOT1, we have isolated Hot1-defective mutants and examined their ability for fork blocking at RFB. Amongst 23 isolated mutants, four were found to be defective in both abilities. Genetic analysis of the mutants reveals that a single mutation, named fob1 (fork blocking less), is responsible for the defects in both abilities. The FOB1 gene is located on chromosome IV and has no homology with any other genes listed in DNA data banks. Conclusion: The pleiotropic effect of the fob1 mutation suggests that homologous recombination at HOT1 is closely linked with DNA replication fork blocking event at RFB.

The master control gene for morphogenesis and evolution of the eye.

Abstract: The human Aniridia, the murine Small eye, and the eyeless mutations of Drosophila affect homologous (Pax-6) genes that contain both a paired- and a homeobox. By ectopic expression of these genes, functional eyes can be induced on the legs, wings, and antennae of the fly, indicating that eyeless (Pax-6) is the master control gene for eye morphogenesis. The finding of Pax-6 from flatworms to humans suggests that eyeless is a universal master control gene and that the various types of eyes in the various animal phyla may have evolved from a single prototype.

Essential and non-redundant roles of p48 (ISGF3 gamma) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies.

Abstract: Background: Interferons (IFNs) are a class of cytokines which confer cellular resistance against viral infections. Type I (IFN-α and -β) and type II (IFN-γ) IFNs utilize distinct receptors, the stimulation of which results in the induction of downstream target genes. These target genes usually contain within their promoter region an IFN responsive element, termed ISRE (IFN stimulated response element) which binds a heterotrimeric transcription factor, ISGF3 (IFN-stimulated gene factor 3) consisting of p48 (ISGF3 γ), Stat1 (Signal transducers and activators of transcription-1; α or β), and Stat2. The ISRE sequence overlaps with that of IRF-E which binds another IFN-inducible factor, IRF-1 (IFN regulatory factor-1). Results: We generated mice lacking p48 by gene targeting. We show that p48 plays an essential role in both type I and type II IFN responses; activation of IFN-inducible genes and establishment of the antiviral state by IFN-α or -γ are both severely impaired, and ISRE-binding activities induced by both IFNs are absent in the p48-negative embryonic fibroblasts (EFs). Furthermore, we generated mice deficient for both p48 and IRF-1 and found that at least one IFN-inducible gene is dependent on both factors. Conclusions: p48 and IRF-1 do not perform redundant functions in the cell, but rather complement one another in both type I and II IFN responses.

The shortest path from the surface to the nucleus: RBP-J kappa/Su(H) transcription factor.

Abstract: Communication between cell surface receptors and nuclear transcription factors is of primary importance to multicellular organisms. Since there are numerous molecules involved in this process, their mutual interaction forms complex networks of informational regulation, which is still under extensive investigation. The RBP-J kappa transcription factor interacts directly with the Notch receptor involved in cell lineage commitment, implicating the presence of a uniquely simple communication strategy between the surface receptor and the nucleus.

Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.

Abstract: Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nm (Kd ≈ 21 nm). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 ≈ 24 min). β is more stable in solution (Kd < 60 pm) and on DNA (t1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd ≈ 250 nm) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.

Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model.

Abstract: Background : The inhibition of β-galactosidase expression in glucose–lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose–lactose system. Results : To examine the roles of cAMP and the cAMP receptor protein (CRP) in the glucose effect, the intracellular levels of these factors were determined during diauxic growth in a glucose–lactose medium. We found that the levels of cAMP and CRP in a lactose-grown phase were not higher than those in a glucose-grown phase, although the cAMP levels increased transiently during the lag phase. The addition of exogenous cAMP eliminated diauxic growth but did not eliminate glucose repression. Glucose repression and diauxie were observed in cells which lack cAMP but produce a cAMP-independent CRP. In addition, inactivation of the lac repressor by the disruption of the lacI gene or the addition of IPTG, eliminated glucose repression. Conclusion : We conclude that the repression of β-galactosidase expression by glucose is not due to the reduction of the cAMP-CRP level but due to an inducer exclusion mechanism which is mediated by the phosphoenolpyruvate-dependent sugar phosphotransferase system.

Mash1 promotes neuronal differentiation in the retina.

Abstract: Background: Mash1, a mammalian homologue of Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurones. Whereas Mash1 is expressed during retinal development, no apparent abnormalities were found during embryogenesis as well as at birth in Mash1-null retina, suggesting that early differentiating cells such as ganglion, amacrine and cone cells develop normally. Because Mash1-null mice die soon after birth, their postnatal development cannot be examined in vivo. Thus, it remains to be determined whether or not Mash1 functions in postnatal development of retina. Results: Here, Mash1 roles in postnatal development of retina was examined by using retinal explant that develops like in vivo retina. Without Mash1, differentiation of late appearing cells such as rod, horizontal, and bipolar cells was delayed and the final number of bipolar cells was significantly reduced. In contrast, vimentin-positive cells (probably Muller glial cells) were increased in Mash1-null retina. Conclusions: These results provide evidence that Mash1 promotes neuronal differentiation during retinal development and is essential for proper ratios of retinal cell types.

Vertebrate non-receptor protein-tyrosine kinase families.

Abstract: Many non-receptor protein–tyrosine kinases (PTKs) function as subunits of receptors, either receptors with or without intrinsic PTK catalytic activity of their own. There are currently at least 33 known vertebrate genes that encode non-receptor PTKs. These can be divided into nine families: Abl, Fes/Fer, Syk/Zap70, Jak, Tec, Fak, Ack, Src, and Csk. Four additional non-receptor PTKs (Rlk/Txk, Srm, Rak/Frk, and Brk/Sik) do not appear to belong to any of the defined families. Here we review current knowledge of the general roles of non-receptor PTKs, as well as the characteristic features and functions of each family and its family members.

The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination.

Abstract: Background: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome-mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants. Results: We have purified the E. coli SbcCD protein from an overexpressing strain and have shown that it has an ATP-dependent DNA double-strand exonuclease activity. Co-purification of nuclease with protein, antibody inhibition and the absence of activity in extracts lacking the sbcCD genes confirm that the activity is intrinsic to SbcCD. The purified protein also has an ATP-independent single-strand DNA endonuclease activity. Conclusions: We have shown that sbcCD encodes a nuclease. The purified protein has double-strand DNA exonuclease and single-strand DNA endonuclease activities. We propose that SbcCD cleaves secondary structures formed at replication forks and that the broken forks can be repaired by homologous recombination.

Schizosaccharomyces pombe gad7+ encodes a phosphoprotein with a bZIP domain, which is required for proper G1 arrest and gene expression under nitrogen starvation

Abstract: Background: Fission yeast cells arrest at G1 phase when starved of nitrogen. The molecular mechanism that ensures this arrest is poorly understood. We took a genetic approach to this problem. Results: The fission yeast gad7-1 mutant failed to arrest at G1 when starved of nitrogen, and was poor in mating and sporulation. The gad7 gene was cloned by complementation. The deduced gad7 gene product was a bZIP protein of 566 amino acids, which could bind to the CRE (cAMP response element) sequence in vitro. Disruption of gad7 resulted in the same phenotypes as gad7-1. Expression of ste11, which encodes a key transcription factor for sexual development, was not inducible in the disruptant. Gad7 was co-immunoprecipitated with another bZIP protein Pcr1, suggesting that the two proteins form a heterodimer in vivo. Gad7 was phosphorylated, and the state of its phosphorylation appeared to be modified in pka1Δ or wis1Δ cells. Conclusions: Gad7, a CRE-binding protein that cooperates with Pcr1, is required for proper G1 arrest and gene expression under nitrogen starvation. Gad7 is a phosphoprotein, whose activity may be regulated by protein kinases including the cAMP-dependent protein kinase (Pka1) and Wis1 osmosensory MAP kinase kinase.

SH2 and SH3‐containing adaptor proteins: redundant or independent mediators of intracellular signal transduction

Abstract: Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein-protein interactions between proline-rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3-containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.

RNA-dependent association of the Drosophila maleless protein with the male X chromosome.

Abstract: Background: Dosage compensation results in equivalent X-linked gene expression in males (XY) and females (XX). In Drosophila, both X chromosomes are active in females, and the single male X must double its transcriptional activity to allow male development. Four proteins (encoded by the male-specific lethal genes) are required for dosage compensation and associate with the X chromosome in males but not in females. Results : In this report, we focus on the maleless (MLE) protein. The MLE protein sequence contains motifs common to members of a family of RNA-dependent ATPases. We have found that association of MLE with the male X chromosome is RNase sensitive, and that mutations in the ATPase motifs affect MLE function. Overexpression of MLE or its carboxyl terminus, which includes glycine-rich repeats, reveals an RNase-sensitive affinity for all chromosome arms. Conclusions: Our results suggest that nascent transcripts or a hypothetical RNA component of chromatin play a critical role in the biochemical mechanism of dosage compensation. The potential relationship between interaction with RNA and transcriptional control of the X chromosome suggests that the mechanism of dosage compensation is distinct from classical models for transcriptional activation.

Rab3A small GTP-binding protein in Ca2+ -dependent exocytosis

Abstract: There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in Ca2+-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.

NAP‐I is a functional homologue of TAF‐I that is required for replication and transcription of the adenovirus genome in a chromatin‐like structure

Abstract: Background: For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor-I (TAF-I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF-I also stimulates transcription from the Ad core DNA. Results: Using mutant TAF-I proteins, we have demonstrated that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease has revealed that TAF-I causes a structural change in the Ad core. TAF-I has been shown to have significant amino acid similarity to nucleosome assembly protein-I (NAP-I), which is involved in the formation of the chromatin structure. We have shown that TAF-I can be substituted by NAP-I in the activation of the cell-free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF-I in NAP-I are required for the maximal TAF-I activity of NAP-I. Furthermore, TAF-I has been shown to have NAP-I activity, and the acidic region of TAF-I is required for this activity. Conclusions: Since TAF-I causes the structural change of the Ad core and thereby activates transcription, TAF-I is thought to be one of the proteins which is involved in chromatin remodeling. NAP-I is structurally related to TAF-I and functionally substitutes for TAF-I. Furthermore, TAF-I has NAP-I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed.

Regulation of IFN‐α/β genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN‐α/β

Abstract: Background: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN-stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN-stimulated response elements) to activate IFN-inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3γ) also binds to virus-inducible elements in the IFN-α/β genes, suggesting a potential new role of p48 in IFN production. Results: Primary cells from mice with a targeted disruption of the p48 gene show severe defects in virus-induced IFN-α/β gene expression. A similar defect was also observed in cells lacking type I IFNR or Stat1, further demonstrating the role of IFN signalling in the induction of these IFN genes. ISGF3 in fact binds to the virus-inducible elements within the IFN-α/β promoters. We also provide evidence showing that these elements are additionally controlled by an unidentified factor(s) which presumably triggers the primary phase of IFN gene induction. Conclusions: Our results demonstrate that the IFN signal transducing complex ISGF3 plays a crucial role in IFN production and suggest that ISGF3 may participate directly in the activation of IFN-α/β promoters. This dual function of ISGF3 may insure the efficient operation of this cytokine system in the host defence.

Enhancer analysis of the mouse HNF‐3β gene: regulatory elements for node/notochord and floor plate are independent and consist of multiple sub‐ elements

Abstract: Background: Axial pattern formation in vertebrate embryos depends on signals from the node and, later, from the notochord and floor plate. Previous studies have shown that HNF-3β, a member of the winged-helix transcription factor family, plays key roles in the development of all three organizing centres. Results: Enhancer analysis of HNF-3β has therefore been performed using lacZ reporter gene constructs in transgenic mouse embryos. This has led to the identification of independent node/notochord and floor plate enhancers, at positions far upstream (−15 to −14 kb) and downstream (+6 to +11 kb) of the transcription start site, respectively. The node/notochord enhancer activity has been localized to a 520 bp fragment. Floor plate gene expression requires a combination of two separate fragments of the enhancer. Deletion analysis of one of these fragments (400 bp) has identified subregions required for the initiation and the maintenance of floor plate expression, and for the suppression of ectopic expression within the neural tube. Conclusion: We conclude that HNF-3β gene expression in the node/notochord and in the floor plate are controlled through different enhancers, which consist of positive and negative elements.

Histone-like protein HU as a specific transcriptional regulator: co-factor role in repression of gal transcription by GAL repressor.

Abstract: Background: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo Results: We have purified a protein that restored the expected GalR-mediated repression for the in vitro system and have identified this factor to be the bacterial histone-like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, d-galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation. Conclusion: We propose that HU, acting as a co-factor, brings about the GalR-mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.

Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli.

Abstract: Background The major cold-shock protein in Escherichia coli is CspA, a 7.4 kDa protein. A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE. The expression of cspB, unlike the other homologues, is cold-shock inducible like cspA Results: We examined the cold-shock induction of CspA and CspB at various temperatures. The cspA induction is observed by temperature shift from 37 to 30 °C and high levels of CspA production are observed between 24 and 10 °C. In contrast, CspB production occurs only by temperature shift to below 20 °C, with maximum induction at 15 °C. Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension. Conclusions: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E. coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature. The cspB promoter shows sequence similarity to the cspA promoter. Furthermore, both cspA and cspB mRNAs have unusually long 5′ untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures. These features are considered to contribute to the nature of the thermostats for cspA and cspB

Subcellular localization of proteins governing the proteolytic activation of a developmental transcription factor in Bacillus subtilis

Abstract: BACKGROUND Spore formation in Bacillus subtilis takes place in a sporangium consisting of two compartments called the forespore and the mother cell. Late in development, when the forespore is wholly contained within the mother cell, gene transcription is coordinated between the compartments by an intercellular signal transduction pathway. This pathway operates at the level of proteolytic processing of the proprotein precursor (pro-sigma K to the mother-cell transcription factor sigma K. The conversion of pro-sigma K to sigma K is governed by the putative processing enzyme SpoIVFB and its negative regulator SpoIVFA, which are produced in the mother cell. RESULTS We used fluorescence microscopy in conjunction with antibodies against SpoIVFA and SpoIVFB and a fusion of SpoIVFB to the Green Fluorescent Protein from Aquorea victoria to visualize these proteins in the sporangium. Both proteins were found to co-localize with the forespore region of the sporangium, a finding consistent with the idea that SpoIVFA and SpoIVFB, which are inferred to be integral membrane proteins, are located in the mother cell membrane that surrounds the forespore. CONCLUSIONS We conclude that SpoIVFA and SpoIVFB are situated at the boundary between the forespore and the mother cell, at which location SpoIVFB could be activated by a signalling protein produced in the forespore.

Mouse MCM proteins: complex formation and transportation to the nucleus

Abstract: Background: The members of the MCM protein family, including MCM2, MCM3, Cdc21, CDC46, Mis5 and CDC47, are considered to be involved in the control of a single round of DNA replication during S phase in eukaryotes. They bind to chromatin during G1 and detach from it during S phase as if they license the chromatin to replicate. However, unlike the originally proposed ‘licensing factor’ and the budding yeast homologues, mammalian MCM2 and P1MCM3 proteins appeared to be localized in the nucleus during the interphase. Results: We purified mCdc21 and its associated proteins from mouse cell extract by anti-mCdc21 immunoaffinity chromatography. Three proteins which co-purified with mCdc21 were identified as mCDC47, mMis5 and mMCM2, all were MCM proteins. Glycerol gradient centrifugation analysis showed that all the mouse MCM proteins were detected at 450–600 kDa, an indication of the sum of their calculated molecular weights from their amino acid sequences. mCdc21 was displaced from replicated chromatin in a similar way to P1MCM3 and MCM2 during S phase. Among the six mouse MCM proteins, only mMCM2 and mP1MCM3 showed nuclear localization when overexpressed in COS cells. Conclusions: We conclude that in the mouse, six MCM proteins form a single protein complex of molecular weight 450–600 kDa, which may enter the nucleus by nuclear localization signals in the mMCM2 and mP1MCM3 subunits.

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo

Abstract: Background: V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this ‘12/23 rule’ regulates cleavage, joining, or both. Results: We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo. Conclusions: These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo.

The HGF receptor family: unconventional signal transducers for invasive cell growth

Abstract: The HGF receptor family includes tyrosine kinases encoded by three oncogenes: MET, SEA and RON. The members of this gene family share a unique functional feature: they mediate cell dissociation and motility ('scattering') in physiological conditions, and invasiveness in their activated versions. The Met, Ron and Sea receptors display a distinctive signal transduction behaviour. Unlike conventional growth factor receptors, their cytoplasmic tails contain a multifunctional docking site. Upon autophosphorylation, this sequence simultaneously binds and activates multiple SH2-containing transducers, including Ras and PI 3-kinase. A deregulated activation of this 'supersite' triggers a dramatic pleiotropic signal which is responsible for invasive cell growth.

The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis

Abstract: BACKGROUND: Protein phosphatase 2A (PP2A) holoenzymes have a trimeric structure, consisting of a catalytic subunit C and two regulatory subunits A (PR65) and B (PR55). In fission yeast the C subunits, being 80% identical to their mammalian counterparts, are essential for viability and negatively regulate the entry into mitosis. Genetic analyses in budding yeast and Drosophila show that the regulatory subunits are implicated in chromosome segregation, cell morphogenesis and/or cytokinesis. RESULTS: We isolated fission yeast genes paa1+ and pab1+ encoding the regulatory subunits PR65 and PR55, respectively. Gene disruption showed that the paa1+ gene was essential for viability while pab1+ was not required at 26-33 degrees C. Microtubule and actin distributions were anomalous in gene disrupted delta paa1 cells which were incapable of forming a polarized cell shape. Gene disrupted delta pab1 cells were pear- or round-shaped, and lost the polar distributions of actin and microtubules. In addition, delta pab1 cells were defective in cell wall synthesis and sporulation at permissive temperatures. At restrictive temperatures, delta pab1 cells showed an osmoremedial temperature-sensitive phenotype and delayed in cytokinesis. However, chromosome segregation was normal. CONCLUSION: Fission yeast PP2A regulatory subunit plays a critical role in cell morphogenesis, probably through regulation of the cytoskeletal network and cell wall synthesis.

Communication between homologous chromosomes: genetic alterations at a nuclease-hypersensitive site can alter mitotic chromatin structure at that site both in cis and in trans

Abstract: Background: In vegetatively growing diploid strains of the yeast Saccharomyces cerevisiae, homologous chromosomes appear to be paired via multiple interstitial interactions, likely as a regular feature of the diploid lifestyle. We have previously suggested that this pairing is guided by direct physical interactions between intact DNA duplexes in nuclease-hypersensitive regions and that homology is sensed directly at the DNA level. Results: As a first test of this idea we have examined the level of DNase I sensitivity at a prominent nuclease-hypersensitive site in mitotic chromatin in strains that are either homozygous or heterozygous for a pair of alleles at this site. We find that the degree of nuclease sensitivity at this site on a given (maternal or paternal) chromosome can vary depending upon whether the homologue carries the same allele or the different allele. The data are suggestive that nuclease sensitivity is higher in the former case than in the latter, as though nuclease hypersensitivity might be increased when the two alleles match as compared to when they do not. Conclusions: Formally, these observations suggest that homologous chromosomes can communicate via a mechanism that senses the status of the assayed nuclease-hypersensitive site with resultant changes in chromatin structure at that site. The observed pattern of effects is fully compatible with direct physical interactions between homologues at nuclease-hypersensitive regions, but alternative scenarios also can be envisioned. Since DNase I hypersensitive sites occur in many important regions of chromosomes, homology-dependent interactions involving such regions could potentially affect diverse processes including gene expression (e.g. transvection), chromosome organization, domain structure, and/or DNA replication patterns.

Processing of the Nedd2 precursor by ICE‐like proteases and granzyme B

Abstract: Background: The Nedd2/Ich-1 protein belongs to a growing family of mammalian cysteine proteases similar to interleukin-1β converting enzyme (ICE). Because of their similarity to the Caenorhabditiselegans cell death protein CED-3, the ICE-like proteins are thought to play a key role in the execution of apoptosis. The active form of ICE is a tetramer consisting of two heterodimers (p20 + p10)2 derived from the cleavage of the pro-enzyme. Results: In the present communication we show that the p51 Nedd2 precursor (pro-Nedd2) is also cleaved into p20-like (p19) and p10-like (p12) subunits by extracts prepared from cultured cell lines. Extracts from apoptotic NIH-3T3 cells but not normal growing NIH-3T3 cells also contained pro-Nedd2 cleaving activity. The processing of pro-Nedd2 by cell extracts was inhibited by characteristic inhibitors of ICE-like proteases. Additionally we show that pro-Nedd2 (p51) can be processed in vitro by active CPP32 and ICE, and to a lesser extent by Mch2 and Nedd2. Granzyme B, a serine protease required for cytotoxic T lymphocyte (CTL) mediated killing of target cells, also cleaved pro-Nedd2 to p19 + p12 subunits. Conclusions: Our observations suggest that Nedd2 activation requires cleavage by one or more ICE-like proteases that lie upstream in the proteolytic cascade. Cleavage of pro-Nedd2 by granzyme B indicates that Nedd2 may be one of the downstream effectors in the CTL-mediated killing of target cells.

MutT-related error avoidance mechanism for DNA synthesis.

Abstract: Mutator mutants that show an increased frequency of spontaneous mutation have led to the elucidation of the multiple pathways of spontaneous mutagenesis. 8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. MutT protein of Escherichia coli and related mammalian enzymes specifically degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of transversion mutation. The gene encoding the human enzyme, designated MTH1 (for mutT homologue 1), maps to chromosome 7p22. These proteins may be responsible for genomic stability.