bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)
Authors: Hromockyj Alexander E. , Tucker Stephanie C. , Maurelli Anthony T.
Published: October 2006

virR is the central regulatory locus required for coordinate temperature‐regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI‐Accl DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique Hpal restriction site resulted in a loss of VirR+ activity. The vir R ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone‐like protein, H‐NS. Based on the predicted amino acid sequence of E. coli H‐NS, only a single conservative base‐pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNATyr. These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.
Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P‐450soy and ferredoxinsoy (soyB) from Streptomyces griseus
Authors: Trower M. K. , Lenstra R. , Omer C. , Buchholz S. E. , Sariaslani F. S.
Published: October 2006

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P‐450, designated cytochrome P‐450soy A DNA segment carrying the structural gene encoding P‐450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P‐450soy was compared with that for P‐450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe‐4S] ferredoxin (soyB), named ferredoxinsoy, was identified.
Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene
Authors: Merino Enrique , Balbás Paulina , Recillas Felix , Becerril Baltazar , Valle Fernando , Bolivar Francisco
Published: October 2006

Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5’‐terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5′‐terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.
Osmostress‐induced changes in yeast gene expression
Authors: Varela Joäo C. S. , van Beekvelt Catelijne , Planta Rudi J. , Mager Willem H.
Published: October 2006

When Saccharomyces cerevisiae cells are exposed to high concentrations of NaCI, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCI) by a previous treatment with 0.7 M NaCI, but not by a previous heat shock. Two‐dimensional analysis of [3H]‐leucine‐labelled proteins from salt‐shocked cells (0.7 M NaCt) revealed the elevated rate of synthesis of nine proteins, among which were the heat‐shock proteins hsp12 and hsp26. Northern analysis using gene‐specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol‐3‐phos‐phate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso‐osmolar NaCI concentration (0.7 M).
Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue‐specific adhesive properties
Authors: Marklund Britt‐lnger , Tennent Jan M. , Garcia Elisa , Hamers Anja , Baga Monika , Lindberg Frederik , Gaastra Wim , Normark Staff an
Published: October 2006

Escherichia coli strains bind to Galα1‐4Gal‐containing glycolipids via P pili‐associated G‐adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G‐alleles. We suggest that the Class I papG‐allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G‐adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual paplprs genes have occurred. We propose that the retention and spread of pap prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.
Identification of a novel large extrachromosomal DNA (LED) in the Trypanosomatidae
Authors: Wagner Wilma , So Magdalene
Published: October 2006

We have identified a novel 75 kbp large extrachromosomal DNA (LED) which is stably maintained during developmental conversion of Trypanosoma cruzi. It has a covalently closed circular conformation and is not derived from the kinetoplast network. In all T. cruzi strains analysed, LED contains 18S rRNA and spliced leader (sl) sequences. LED from the T. cruzi Y strain contains a minimum of 15 copies of the sl repeat arrayed in a head‐to‐tail configuration and 50 copies of a 196 bp repeat. LED is also present in Trypanosoma dionisii (subgenus Schizotrypanosoma) and in other members of the family Trypanosomatidae. LED from different T. cruzi strains and from other members of the Trypanosomatidae differ in their content of large ribosomal subunit rRNA sequences and the 196 bp repeat. The presence of LED in four evolutionarily distant trypanosomatid species suggests that it plays an important rote in the biology of these parasites.
The effects of 5′‐capping, 3′‐polyadenylation and leader composition upon the translation and stability of mRNA in a cell‐free extract derived from the yeast Saccharomyces cerevisiae
Authors: Gerstel Birgit , Tuite Mick F. , McCarthy John E. G.
Published: October 2006

A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell‐free extracts. The system was used to examine the influence of 5′‐capping, 3′‐polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat‐shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap‐independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.
Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity
Authors: Drefus Lawrence A. , Urban Robert G. , Whipp Shannon C. , Slaughter Clive , Tachias Kathy , Kupersztoch Yankel M.
Published: October 2006

The methanol‐insolouble heat‐stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino‐terminal residue, Ser‐24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys‐33 and Cys‐71 and between Cys‐44 and Cys‐59. Oligonucleotide‐directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys‐44—Cys‐59 bond contains at least two amino acids (Arg‐52 and Asp‐53) required for STQ toxic activity.
Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB‐dependent proteins
Authors: Goldberg Marcia B. , Boyko Stephanie A. , Butterton Joan R. , Stoebner Janice A. , Payne Shelley M. , Calderwood Stephen B.
Published: October 2006

IrgA is an iron‐regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the EI Tor biotype, three non‐O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to IrgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron‐regulated outer membrane protein of V. cholerae, we determined the amino‐terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two EI Tor strains of V. cholerae had a major iron‐regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in IrgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB‐dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol‐substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB‐like protein in V. cholerae remain to be determined.
DNA curving and bending in protein–DNA recognition
Authors: Harrington Rodney E.
Published: October 2006

Most biological events are regulated at the molecular level by site‐specific associations between specialized proteins and DNA. These associations may bring distal regions of the genome into functional contact or may lead to the formation of large multisubunit complexes capable of regulating highly site‐specific transactional events. It is now believed that sequence‐specific protein–DNA recognition and the ability of certain proteins to compete for multiple binding sites is regulated at several levels by the local structure and conformation of the binding partners. These encompass the micro structure of DNA, including its curvature, bending and flexing as well as conformational lability in the DNA‐binding domains of the proteins. Possible mechanisms for binding specificity are discussed in the context of specific nucleoprotein systems with particular emphasis given to the roles of DNA conformations in these interactions.
Construction of chimaeric genes for mapping a surface‐exposed epitope on the pilus of non‐typable Haemophilus influenzae strain M37
Authors: Palmer Katherine L. , Munson Robert S.
Published: October 2006

A murine monoclonal antibody (mAb), designated 3H12, reacts with a surface‐exposed conformational epitope on the pilus of non‐typable Haemophilus influenzae strain M37. This antibody does not recognize the related pilus from H. influenzae type b, strain MinnA. Although mAb 3H12 does not recognize strain M37 pilin on Western blots, mAb 3H12 recognizes the recombinant M37 pilin protein expressed by Escherichia coli. In order to map the epitope recognized by mAb 3H12, we constructed a series of chimaeric genes. The chimaeric genes were expressed in E. coli and the chimaeric proteins characterized with respect to their reactivity with mAb 3H12. Residues between 37 and 100 of the M37 pilin protein are essential for the expression of the mAb 3H12 epitope. Residues in the carboxyl half of the M37 protein enhance the reactivity of mAb 3H12 when expressed in the presence of residues 37–100. Construction of chimaeric genes may provide a general methodology for mapping of conformational epitopes expressed by one of a related pair of proteins.
Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymerase
Authors: Zou Chao , Fujita Nobuyuki , Igarashi Kazuhiko , Ishihama Akira
Published: October 2006

The C‐terminal region (amino acid residues 236–329) of the Escherichia coli RNA polymerase α subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C‐terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant a subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP–CRP. DNA sequence determination of several ‘Lac‐’ mutant rpoA genes revealed that all had mutations clustered within a short segment near the C‐terminus of α between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).
Expression of a functional neisserial fbp gene in Escherichia coli
Authors: Berish Sally A. , Chen Cheng‐Yen , Mietzner Timothy A. , Morse Stephen A.
Published: October 2006

The ability to acquire iron from a human host is a major determinant in the pathogenesis of Neisseria gonorrhoeae and Neisseria meningitidis. Pathogenic Neisseria spp. do not synthesize siderophores and instead express a receptor‐mediated, high‐affinity iron acquisition system in the iron‐restricted environment of its host. A ferric‐iron‐binding protein (Fbp) of Neisseria spp. is also iron‐regulated and may play a central role in this novel iron‐uptake system. To define the physical properties of Fbp further, we used polymerase chain reaction to synthesize DNA fragments containing the fbp structural gene with and without the sequence encoding the Fbp leader peptide. These fragments were ligated into pUC13 to create in‐frame fusions with the alpha peptide of lacZ. The expression of Fbp was under the control of the lacZ promoter. Both fusion clones produced Fbp in large amounts, facilitating the purification of quantities of Fbp sufficient for elucidating the biochemical, immunologic, and functional properties of this protein.
Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element
Authors: Fetherston Jacqueline D. , Schuetze Paul , Perry Robert D.
Published: October 2006

The pigmentation. (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm‐ mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm‐ mutant, KIM6. Yersinia pseudo‐tuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA‐LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.
Frameshifting in the expression of the Escherichia coli trpR gene
Authors: Benhar Itai , Miller Chaya , Engelberg‐Kulka Hanna
Published: October 2006

The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site‐specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N‐terminus of the +1 frame product and that of the known 0 frame product are identical but that their C‐termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.
The glucose kinase gene of Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression
Authors: Angell S. , Schwarz E. , Bibb M. J.
Published: October 2006

Mutants (glk) of Streptomyces coelicolor A3(2) that are resistant to the non‐utilizable glucose analogue 2‐deoxyglucose are deficient in glucose kinase activity, defective in glucose repression, and usually unable to utilize glucose. A 2.9 kb Bcll fragment, previously shown to restore a wild‐type phenotype to a glk deletion mutant that lacks the entire segment, contains two complete open reading frames that would encode proteins of 20.1 kDa (ORF2) and 33.1 kDa (ORF3). ORF3 is transcribed from its own promoter, and also from a promoter that initiates transcription upstream of ORF2. A derivative of the temperate phage πC31 containing ORF3 alone restored a wild‐type phenotype when used to lysogenize the deletion mutant. The product of ORF3 is homologous to members of a family of repressor proteins encoded by xylR In Bacillus subtilis and Lactobacilius pentosus, and by nagC in Escherichia coli. Although this might suggest that ORF3 encodes a positive activator for glucose kinase, rather than the enzyme itself, ORF3 restored the ability to metabolize glucose to an E. coli glk mutant, and activity gels of cell extracts of E. coli containing ORF3 cloned in the pT7‐7 expression vector demonstrated that the ORF3 product has glucose kinase activity.
Separation of two lipopolysaccharide populations with different contents of O‐antigen factor 122 in Salmonella enterica serovar Typhimurium
Authors: Helander Ilkka M. , Moran Anthony P. , Mäkela P. Helena
Published: October 2006

Lipopolysaccharide (LPS) extraction from smooth‐type Salmonella enterica sv. Typhimurium was carried out with the modified phenol/chloroform/ petroleum ether method (volume ratio 5: 5:8). In this procedure, LPS was precipitated from 90% phenol sequentially with water and acetone to yield LPS‐H2O (minute amounts) and LPS‐Ac (major amounts), respectively. Chemical analyses of the LPS fractions revealed that in the O antigen of LPS‐H2O position C4 of the D‐galactose was extensively glucosylated, corresponding to the O‐antigen factor 122. In LPS‐Ac, this glucosylation was negligible. Inspection of the LPS fractions by sodium dodecyl sulphate polyacryl‐amide gel electrophoresis and silver staining suggested that the glucosylation in LPS‐H2O was present only in LPS species with a chain length higher than six repeating units.
Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph
Authors: Hinshelwood S. , Stoker N. G.
Published: October 2006

Histidine‐requiring auxotrophs of Mycobacterium smegmatis were isolated following N‐methyl‐N′‐nitro‐ N‐nitrosoguanidine treatment. One of these mutants, his 5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial‐specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.
Mechanisms of the cytopathic action of actin‐ADP‐ribosylating toxins
Authors: Aktories K. , Wegner A.
Published: October 2006

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP‐ribosylate actin monomers. Toxin‐induced ADP‐ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP‐ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.
cis‐Unsaturated fatty acids specifically inhibit a signal‐transducing protein kinase required for initiation of sporulation in Bacillus subtilis
Authors: Strauch Mark A. , de Mendoza Diego , Hoch James A.
Published: October 2006

The initiation of sporutation in Bacillus subtilis is controlled by the Spo0A transcription factor which is activated by phosphorylation through a phosphorelay mechanism that is dependent upon the activity of one or more protein kinases. The enzymatic activity of one of these protein kinases, KinA, was found to be inhibited in vitro by certain fatty acids. The most potent inhibitors have at least one unsaturated double bond in the cis configuration and a chain length of 16–20 carbon atoms. Homologous isomers with a trans double bond are not inhibitory. Saturated straight‐ or branched‐chain fatty acids are either much weaker inhibitors or have no effect. The inhibitors prevent autophosphorylation of KinA and are non‐competitive with ATP. B. subtilis phospholipids were found to contain at least one as yet unidentified type of fatty acid that, when present in an unesterified form, inhibited KinA. The results suggest that the concentration of a specific unsaturated fatty acid may act as a signal linking the initiation of sporulation to the status of membrane synthesis and septation or some other specific membrane‐associated activity.
Cloning and characterization of a gene encoding flagellin of Listeria monocytogenes
Authors: Dons L. , Rasmussen O.F. , Olsen J.E.
Published: October 2006

The gene, flaA, encoding the flagellin protein of Listeria monocytogenes (strain 12067) has been isolated from an expression library in Escherichia coli using a flagellin‐specific monoclonal antibody. DNA sequence analysis of a positive clone revealed the presence of an open reading frame of 287 amino acid residues with a calculated molecular mass of 30.4 kDa. Comparison of this sequence with flagellins from other bacteria showed a significant degree of homology in both the N‐ and C‐terminal parts of the protein. The flagellin mRNA was determined to be 1 kb in size, which is the expected size for a monocistronic mRNA, and the temperature‐dependent expression of flagellin was found to be regulated at the transcriptional level. Southern blot analysis, using the flagellin gene as probe, indicated that L. monocytogenes can be divided into two groups. These groups correspond to the flagellar antigens AB and ABC, respectively, as well as to the two types of L. monocytogenes based on the DNA sequence of the listeriolysin gene.
Gene disruption identifies a 290 kDa cell‐surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii
Authors: McNab R. , Jenkinson H. F.
Published: October 2006

The C‐terminal coding region of the gene (denoted csh A) encoding a high‐molecular‐mass (290 kDa) cell‐surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3′ end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell‐surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus‐anchored cell‐surface protein determinant of hydrophobicity and coaggregation in S. gordonii
Determinants of an unusually stable mRNA in the bacterium Myxococcus xanthus
Authors: Romeo Joseph M. , Zusman David R.
Published: October 2006

Myxococcus xanthus is a Gram‐negative bacterium which has a complex life cycle that includes development (fruiting body formation). The gene for myxo‐bacterial haemagglutinin, mbhA, is developmentally regulated and highly expressed. In this report we show that the mbhA mRNA is exceptionally stable for a prokaryotic organism, exhibiting a chemical half life (t1/2) of 150 min at 18 h of development. The mbhA mRNA was not stable in negetatively growing cells nor was it stable when expressed in Escherichia coli. We have used site–directed mutagenesis of the mbhA gene to analyse some of the determinants which mediate the stability of the mbhA transcript. Sequences within the 3′‐untranslated region (3′‐UTR) were found to be crucial for mRNA stability. This region of mRNA can potentially form an extremely stable stem‐loop structure immediately adjacent to the translational stop codon. A deletion within this region caused a 10‐fold increase in the decay rate of the transcript. Furthermore, conditions which were associated with reduced mbhA translation or mutations that caused premature termination of translation drastically reduced mRNA stability even in the presence of the wild type 3′‐UTR. These results suggest that a significant aspect of mbhA mRNA stability involves a synergistic interaction of the translational machinery with sequence elements within the 3′‐UTR.
Identification of functional regions of the positively acting regulatory gene amdR from Aspergillus nidulans
Authors: Parsons Linda M. , Davis Meryl A. , Hynes Michael J.
Published: October 2006

The amdR (intA) regulatory gene of Aspergillus nidulans encodes a 765‐amino‐acid polypeptide which determines the omega‐amino acid induction of at least five structural genes. The AmdR polypeptide contains a potential Zn(ll)2Cys6 DNA‐binding motif which has been shown to be present in the N‐terminal region of a large number of fungal activator proteins. In vitro mutagenesis of the fourth cysteine of this motif abolishes AmdR function as shown by loss of complementation of an amdR− mutation and by the AmdR− phenotype of a mutant gene replacement strain. Studies using constructs in which the proposed AmdR DNA‐binding motif is replaced with that from another activator, FacB, shows that induction is independent of DNA‐binding specificity and that sequences in the C‐terminal region of AmdR are activation domains. Sequencing of several amdR mutant alleles which affect activation and/or induction, together with studies of deletion constructs indicate that changes in the conformation of the protein determines its activity and that this is modulated by inducers.
The 5′ to 3′ exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae
Authors: Díaz Asunción , Lacks Sanford A. , López Paloma
Published: October 2006

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5′ to 3′ exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild‐type polA gene in the chromosome unless the 5′ to 3′ exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5′ to 3′ exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.
Identification, genetic analysis and characterization of a sugar‐non‐specific nuclease from the cyanobacterium Anabaena sp. PCC 7120
Authors: Muro‐Pastor A. M. , Flores E. , Herrero A. , Wolk C. P.
Published: October 2006

A nuclease that could be recovered from the supernatant of cultures, as well as from cell‐free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA‐containing SDS‐polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29650, presented a presumptive signal peptide in its N‐terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.
Direct visualization of plasmid DNA in bacterial cells
Authors: Eliasson Åsa , Bernander Rolf , Dasgupta Santanu , Nordström Kurt
Published: October 2006

The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase‐fluorescence microscopy of DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.
Purification and functional characterization of the KdgR protein, a major repressor of pectinolysis genes of Erwinia chrysanthemi
Authors: Nasser W. , Reverchon S. , Robert‐Baudouy J.
Published: October 2006

The phytopathogenicity of the enterobacterium Erwinia chrysanthemi chiefly results from its capacity to degrade pectin, which is the major component of plant cell walls. This degradation requires the product of 12 genes which constitute independent transcriptional units. All these genes, including kdgT which encodes the 2‐keto‐3‐deoxygluconate (KDG) transport system, are negatively regulated by the KdgR protein. The E. chrysanthemi KdgR gene was cloned into an expression vector and overexpressed in Escherichia coli. KdgR was then purified to homogeneity by two chromatographic steps as a dimer of approximately 62kDa. Using gel retardation assays, we demonstrated that this purified repressor binds to the 25 bp oligonucleotide (AAAAAAGAAACATTGTTTCATTTGT) present in the kdgT regulatory region. Dimethyl sulphate interference experiments revealed that the repressor interacts with four guanine bases and 10 adenine bases in the two strands of this KdgR box. KDG, an actual inducer of pectinolysis, releases the repressor from the operator complexes, whereas galacturonate, which is the precursor of the actual inducer, does not. These results suggest the existence of a specific interaction between KDG and KdgR protein. This study opens discussion on the relative affinity of the KdgR protein for the different operators of pectinolysis genes which are interpreted in terms of differential regulation.
Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis
Authors: Czaplewski L. G. , North A. K. , Smith M. C. M. , Baumberg S. , Stockley P. G.
Published: October 2006

The arginine‐dependent repressor‐activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCO1, with the highest affinity for protein, is located within the 5′ promoter sequences, whilst the other, argCO2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCO1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCO2in vivo forming a repression loop similar to those observed for the E coli Lac repressor.
Transcription activation at Class I CAP‐dependent promoters
Authors: Ebright Richard H.
Published: October 2006

Catabolite gene activator protein (CAP)‐dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP‐dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter‐proximal subunit of CAP are essential for transcription activation at Class I CAP‐dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP‐DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP‐DNA complex. Amino acids 261, 265, and 270 of the α subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP‐dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP‐dependent promoters requires a direct protein‐protein contact between amino acids 156 to 162 of the promoter‐proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein–protein contact involves amino acids 261 and 265 of the α subunit of RNA polymerase.
Location and orientation of an activating region in the Escherichia coli transcription factor, FNR
Authors: Bell Andrew , Busby Steve
Published: October 2006

We have characterized a number of mutations in fnr that interfere with FNR‐dependent transcription activation at two promoters where the FNR‐binding site is centred around 41½ bp upstream from the transcription start site. The substituted residues in all but one of these FNR mutants are clustered around a presumed surface‐exposed beta‐turn containing G85 which, we suggest, forms an activating region that contacts RNA polymerase at these promoters. Using the‘oriented heterodimers’method described elsewhere, we show that this activating region on the promoter‐proximal subunit of the FNR dimer is sufficient to activate transcription initiation. In contrast, this region is not essential for activation of a third FNR‐dependent promoter where the FNR‐binding site is centred at 61 1/2 bp upstream from the transcription start site. However, a substitution at S73 interferes with FNR‐dependent activation at both this promoter and promoters in which the FNR site is located at 41 1/2 bp from the transcript start, suggesting that FNR may contain a second activating region.
The identification of rofA, a positive‐acting regulatory component of prtF expression: use of an mγδ‐based shuttle mutagenesis strategy in Streptococcus pyogenes
Authors: Fogg George C. , Gibson Carmela M. , Caparon Michael G.
Published: October 2006

Binding of the Gram‐positive pathogenic bacterium Streptococcus pyogenes (group A streptococcus) to respiratory epithelium is mediated by the fibronectinbinding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S. pyogenes. Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis in Escherichia coli using a derivative of transposon mγδ that was modified to contain a streptococcal antibiotic‐resistance gene. Mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectinbinding phenotypes revealed that insertions in a region upstream of prtF abolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designated rofA (regulator ofF). Chromosomal interruption studies using integrationat plasmids together with complementation data from a previous study (VanHeyningen etal., 1993) suggested that rofA acts as a positive trans‐acting regulator of prtF. Construction of prtF‐lacZ fusions indicated that transcription of prtF is constitutive in JRS4 but is regulated in rofA mutants. Analysis of the DNA sequence defined by the rofA insertions revealed a 1495bp open reading frame, whose predicted product (RofA) possessed both a putative helix‐turn‐helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram‐positive surface proteins. Sequences homologous to rofA were found in regulated strains of 5. pyogenes, which suggests that rofA may act as an activator of prtF in response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it constitutively active.
Cloning and characterization of a gene for a 19kDa fibrinogen‐binding protein from Staphylococcus aureus
Authors: Bodén Maria K. , Flock Jan‐Ingmar
Published: October 2006

Staphylococcus aureus has been shown to interact specifically with fibrinogen. Three different extracellular fibrinogen‐binding proteins, two of which have coagulase activity, are produced by S. aureus strain Newman. The role of these fibrinogen‐binding proteins during staphylococcal colonization and infection has not yet been fully elucidated. Here we describe the cloning, sequencing and expression of a gene for a 19kDa fibrinogen‐binding protein. This gene, called fib, encodes a 165‐amino‐acid polypeptide, including a 29‐amino‐acid signal sequence. The recombinant protein, which has an estimated molecular mass of 15.9kDa, bound fibrinogen and was recognized by a polyclonal antiserum against the native Fib protein. Homologies between the Fib protein and the fibrinogen‐binding domain of coagulase suggest that amino acids within this domain are involved in the binding to fibrinogen.
IS231A insertion specificity: consensus sequence and DNA bending at the target site
Authors: Hallet Bernard , Rezsöhazy René , Mahilton Jacques , Delcour Jean
Published: October 2006

In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5′‐GGG(N)5CCC‐3′consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly ‘S’ ‐shaped structure centred on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.
Cleavage of trehalose‐phosphate in Bacillus subtilis is catalysed by a Phospho‐α‐(1–1)‐glucosidase encoded by the treA gene
Authors: Helfert Christine , Gotsche Susann , Dahl Michael K.
Published: October 2006

A 2.5 kb DNA fragment contain a gene encoding a phospho‐α‐(1–1)‐glucosidase (phosphotrehalase), designated treA, was isolated from a Bacillus subtilis chromosomal library by complementation of the tre‐12 mutation. The major TreA activity was found in the cytoplasm. TreA exhibits high sequence similarity to thermostable oligo 1,6 β‐glucosidases of several species and the trehalose‐6‐phosphate hydrolase TreC of Escherichia coli. TreA activity is induced by trehalose and repressed by glucose, fructose or mannitol. Induction by trehalose and repression by glucose are concentration dependent. The highest activity of TreA occurs 90min before the end of the exponential growth phase in crude cell extracts. The enzyme is able to cleave para‐nitrophenyl‐glucopyranoside and trehalose‐6‐phosphate but not trehalose. These results indicate that treA encodes a specific phospho‐α‐(1–1)‐giucosidase which cleaves trehalose‐6‐phosphate in the cytoplasm after transport and phosphorylation of trehalose. The 5′ flanking region of treA contains an open reading frame which was partially sequenced, whose product shows about 40% identity to sucrose Enzyme II of the phospho‐transferase transport system from several organisms.
Variable style of transition between Palaeogene fluvial fan and lacustrine systems, southern Pyrenean foreland, NE Spain
Authors: SÁEZ ALBERTO , ANADÓN PERE , HERRERO MARÍA J. , MOSCARIELLO ANDREA
Published: October 2006

Two Palaeogene fluvial fan systems linked to the south‐Pyrenean margin are recognized in the eastern Ebro Basin: the Cardona–Súria and Solsona–Sanaüja fans. These had radii of 40 and 35 km and were 800 and 600 km2 in area respectively. During the Priabonian to the Middle Rupelian, the fluvial fans built into a hydrologically closed foreland basin, and shallow lacustrine systems persisted in the basin centre. In the studied area, both fans are part of the same upward‐coarsening megasequence (up to 800 m thick), driven by hinterland drainage expansion and foreland propagation of Pyrenean thrusts. Fourteen sedimentary facies have been grouped into seven facies associations corresponding to medial fluvial fan, channelized terminal lobe, non‐channelized terminal lobe, mudflat, deltaic, evaporitic playa‐lake and carbonate‐rich, shallow lacustrine environments. Lateral correlations define two styles of alluvial‐lacustrine transition. During low lake‐level stages, terminal lobes developed, whereas during lake highstands, fluvial‐dominated deltas and interdistributary bays were formed. Terminal lobe deposits are characterized by extensive (100–600 m wide) sheet‐like fine sandstone beds formed by sub‐aqueous, quasi‐steady, hyperpycnal turbidity currents. Sedimentary structures and trace fossils indicate rapid desiccation and sub‐aerial exposure of the lobe deposits. These deposits are arranged in coarsening–fining sequences (metres to tens of metres in thickness) controlled by a combination of tectonics, climatic oscillations and autocyclic sedimentary processes. The presence of anomalously deeply incised distributary channels associated with distal terminal lobe or mudflat deposits indicates rapid lake‐level falls. Deltaic deposits form progradational coarsening‐upward sequences (several metres thick) characterized by channel and friction‐dominated mouth‐bar facies overlying white‐grey offshore lacustrine facies. Deltaic bar deposits are less extensive (50–300 m wide) than the terminal lobes and were also deposited by hyperpycnal currents, although they lack evidence of emergence. Sandy deltaic deposits accumulated locally at the mouths of main feeder distal fan streams and were separated by muddy interdistributary bays; whereas the terminal lobe sheets expand from a series of mid‐fan intersection points and coalesced to form a more continuous sandy fan fringe.
The Significance of the Patient in the Training of Analysts: Observations on Harold Stone's Paper
Authors: MARSHAK MILDRED D.
Published: October 2006

For purposes of comparison the basic requirements of the London Society of Analytical Psychology are listed below:

A candidate may apply for training after 350 hours' analysis covering a period of three years. This analysis continues during training.

Once training starts the candidate is required to analyse two patients provided by the C. G. Jung Clinic, to which payments are made. The patients are analysed under supervision. The supervisor is not the candidate's analyst. The candidate is required to attend supervision once a week.

Seminars are held twice a week for three terms of ten weeks, each year.

There has been no obligation to submit or read to the Society either a case report or a theoretical discussion.

After two years' training the candidate may apply for membership of the Society and is accepted or rejected by a Professional Committee to whom the analyst, supervisor, and seminar leaders report.

Editor]
Critical Notice
Authors: Williams Mary
Published: October 2006

Marie‐Louise von Franz (Ed., with commentary). Translated by R. F. C. Hull and A. S. B. Glover. Aurora consurgens
Book Reviews
Authors: Williams Mary
Published: October 2006

Joseph Campbell: Creative mythology

Eleanor Bertine: Jung's contribution to our time

Michael Balint: The basic fault

Heinrich Racker: Transference and counter‐transference

David Bakan: Disease, pain and sacrifice

Asenath Petrie: Individuality in pain and suffering

Bruno Bettelheim: The empty fortress: infantile autism and the birth of the self

Paul Roazen: Freud: political and social thought

Joel Allison, S. J. Blatt and C. N. Zimet: The interpretation of psychological tests

Charles Rycroft: Anxiety and neurosis

Anthony Storr: Human aggression

Michael Courtenay: Sexual discord in marriage

Jolande Jacobi. Frauenprobleme, Eheprobleme

J. M. Heaton: The Eye; phenomenology and psychology of function and disorder