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Lists the research papers to be published in the succeeding issues of the "European Journal of Biochemistry." "Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers," by P.V. Avdonin and colleagues; "Complete nucleotide sequence of the gene for human C1 inhibitor with an unusually high density of Alu elements," by P.E. Carter and colleagues.

Presents several forthcoming papers published on the December 1, 1983 issue of the "European Journal of Biochemistry." "Purification and Properties of a Thiol Protease From Rat-Liver Nuclei"; Changes in Proteoglycan Compositiion of F9 Teratocarcinoma Cells Upon Differentiation"; "Supercoiling and the Mechanism of Restriction Endonucleases"; Identification of the Gene for DNA Helicase II of Escherichia Coli."

This article presents a list of some of the forthcoming papers in biochemistry to be published in this journal. Some of the papers listed are "The Organization of the Chloroplast DNA in wheat and maize in the region containing the LS Gene," by B. Koller, H. Delius and T.A. Dyer, "Effect of Carbamination on the Buffering Power of Purified Human Hamoglobins A Solutions At Two Temperatures," by M. Castaing, E. Bursaux and C. Poyart, "The Complete Nucleotide of the Chicken Ovotransferrin mRNA," by J.M. Jeltsch and P. Chambon, "The Primary Structure of Hen Ovotransferrin," by J. Williams, T.C. Elleman, I.B. Kington, A.G. Wilkins and K.A. Kuhn.

The article presents the list of research papers related to biochemistry, that will be published in forthcoming issues of the "European journal of Biochemistry." Some of these papers are--'A novel testis-specific 105-kDa protein related to 90-kDa heatshock protein,' by H. Itoh and Y. Tashima, 'Casein kinase II phosphorylates DNA-polymerase-a-DNA-primase without affecting its basic enzymic properties,' by V. Podust, G. Bialek, H. Stembach and F. Grosse, 'Structural and functional aspects of calcium homeostasis in eukaryotic cells,' by D. Pietrobon, F. di Virgilio and T. Pozzan.

Presents a list of biochemistry papers due for publication after the March 1991 issue of the "European Journal of Biochemistry". "Complete structure of the chicken α2(VI) collagen gene"; "Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase".

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322, by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from rnRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper. were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chiorophylle containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed. [ABSTRACT FROM AUTHOR]

Transcripts from histone H5 and α and β chicken globin genes were analysed in total RNA extracted from 2-6-day-old chick embryos Denatured RNA, separated on gels, was transferred to either aminophenylthioether (APT) paper or to nitrocellulose and was detected with 32P-labeIled recombinant DNA probes. Good resolution was achieved with methyl mercury hydroxide gels and aminophenylthioether transfer, allowing estimation of H5 and α and β globin rnRNA sizes. With glyoxal gels and nitrocellulose transfer, α and β globin mRNAs were not resolved, but an increased sensitivity of detection of at least 10-fold was achieved. Levels of H5 and α g]obin rnRNAs increased in parallel over 2-6 days. The appearance of definitive red cells in 6-day embryos was indicated by detection of adult β globin transcripts at this stage. [ABSTRACT FROM AUTHOR]

The experiments reported in this paper lead to the following conclusions: 1. An endonucleolytic activity is associated with the "replication site" in Escherichia coli cells; this endonucleolytic activity attacks the complementary strand of the ψx replicative form DNA only. 2. the viral strand of the ψx-replicative form DNA remains circularly closed during the replication cycle; 3. In the immediate replication product both composing strands are linear; in circularization no strand is preferred; 4. A replication product with both strands closed is transiently found in a structural state which can be, under our experimental conditions, only partially denatured by alkali. Subsequently it is transformed to a typical supercoiled RFI structure. [ABSTRACT FROM AUTHOR]

The published sequence of the Vibrio cholerae genome indicates that, in addition to the genes that encode proteins of known and unknown function, there are 1577 ORFs identified as conserved hypothetical or hypothetical gene candidates. Because the annotation is not 100% accurate, it is not known which of the 1577 ORFs are true protein-coding genes. In this paper, an algorithm based on the Z curve method, with sensitivity, specificity and accuracy greater than 98%, is used to solve this problem. Twenty-fold cross-validation tests show that the accuracy of the algorithm is 98.8%. A detailed discussion of the mechanism of the algorithm is also presented. It was found that 172 of the 1577 ORFs are unlikely to be protein-coding genes. The number of protein-coding genes in the V. cholerae genome was re-estimated and found to be ≈ 3716. This result should be of use in microarray analysis of gene expression in the genome, because the cost of preparing chips may be somewhat decreased. A computer program was written to calculate a coding score called VCZ for gene identification in the genome. Coding/noncoding is simply determined by VCZ > 0/VCZ < 0. The program is freely available on request for academic use. [ABSTRACT FROM AUTHOR]

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts. [ABSTRACT FROM AUTHOR]

From neurohaemal lobes of corpora cardiaca of Locusta migratoria a 5-kDa peptide has been isolated and its sequence established [see the accompanying paper, by Hietter et al. (1990) Eur. J. Biochem. 187, 241-247]. We have designed oligonucleotide probes from the peptide sequence of this molecule and screened a library prepared from mRNA of the neurosecretory cell region of the brain of this insect. Several positive cDNAs were isolated, the combined nucleotide sequences of which predict a large precursor of 145 residues (15770 Da) containing the newly isolated 5-kDa peptide. The peptide is flanked by regions homologous to the A and B chains of the superfamily of insulins. The overall organization of the precursor is as follows: signal peptide/domain homologous to the B chain of insulins/C (connecting)-peptide (corresponding to the newly isolated 5-kDa peptide)/domain homologous to the A chain of insulins. The numbers and relative positions of the cysteines of the Locusta peptide are equivalent to those of the other members of the insulin superfamily and most of the hydrophobic core residues are conserved.

Polymorphism and microheterogeneity of DNA appear today as being capable of having important biological consequences. This paper presents a synthetic view of two major properties of the main known forms of DNA (A, B, alternating B, C, D and Z), namely their molecular electrostatic potentials and steric accessibilities. Variations in these properties are explained in terms of the conformational changes involved and deductions are drawn concerning their influence on the interactive properties of DNA with external agents. [ABSTRACT FROM AUTHOR]

By treatment of Bacillus subtilis MO-101-P spoA- met thr- su- with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B. subtilis MO-101-P spoA- [met-]+ thr- su+44, was isolated. This strain does not suppress phage Φ29 mutant susB47, selected on a B. subtilis strain containing the su+3 suppressor isolated by Georgopoulos. A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. The efficiency of suppression by strain su+44 is about 50%. The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor. [ABSTRACT FROM AUTHOR]

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF-hand superfamily of Ca2+-modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low-molecular-mass, Ca2+-binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J & Beisson, J. (1991) Biol. Cell 71, 217-225]. In this paper we show that these polypeptides are recognized by specific anti-centrin polyclonal antibodies. Their microsequences revealed four distinct N-termini. For one of them, ICL1, N-terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identify with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co-expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism. [ABSTRACT FROM AUTHOR]

Type -I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells to lymphoid origin,k while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN -β is induced in a variety of cells of different origin, while not all of these are able to secrete IFN- α. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A 1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A 1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells. [ABSTRACT FROM AUTHOR]

In this paper we compare the sequences of a segment of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene, isolated from eleven different strains belonging to four species of the fungal order Mucorales, Parasitella parasitica, Absidia glauca, Mucor mucedo (Mucoraceae) and Blakeslea trispora (Choanephoraceae). The segment was obtained by polynucleotide-chainreaction amplification with primers derived from conservative regions of the gene. For the species M. mucedo and P parasitica we have obtained evidence for two different types of HMG-CoA reductase genes by hybridization of genomic DNA with the amplified fragment and by cloning and sequencing of two different fragments. The different genes from one species show a sequence similarity of around 80% at the protein sequence level, whereas sequences of the same type from different species show similarity ranging between 91-96%. The highest similarity was found between the genes of type 1 from B. trispora and M. mucedo, although these species belong to different families. Southern-blot analysis of A. glauca DNA and B. trispora DNA revealed a second copy of the genes. [ABSTRACT FROM AUTHOR]

We constructed an artificial holoprotein as a complex between α globin from human adult hemoglobin and the protoporphyrin IX-Zn(II).The prosthetic group is bound in a single conformation to the apoglobin via a coordinative bond between Zn(II) ion and the proximal histidine (His87).The complex is diamagnetic and does not bind either CO nor O2 thus representing a diamagnetic model of deoxygenated α chains.In the present paper we report extensive resonance assignment in the proton nuclear magnetic resonance spectrum of the Zn-substituted α chains in phospate buffer pH 5.6.A large number of aromatic and aliphatic side chain spin systems were identified in the two-dimensional homonuclear COSY spectra.Based on the assigned resonance of heme substituent protons and their NOE cross-peaks, we assigned the majority of resonances representing the heme pocket ide chains. Using the main-chain-directed assignment strategy, we could establish several continuous patterns of sequential assignment and identify partial or total spin systems for a large number of side chains.The final assignment corresponds to 73% of the amino acids. Analysis of chemical shift of assigned resonances and of nuclear Overhauser enhancement connectivities provides structural information on the global and local tertiary conformation in solution and on the ligand-induced conformational changes. Comparison of observed and calculated ring current shifts enabled us to compare the solution structure with the X-ray crystal structure of α subunits in deoxy and carbonmonoxy hemoglobin. The global tertiary structure of unliganded chains is highly similar to both liganded and unliganded counterparts in the crystalline state. On the distal side of the heme pocket, Va162 is significantly closer to the heme center, in agreement with its conformation in the crystallographic structure.In contrast, the position of the proximal histidine (His87) relative to the heme is clearly more closely related to that in the liganded tetramer in the crystalline state. Comparison of the chemical shift values for the resonances in carbon monoxy and Zn(II)-substituted α chains indicates that the ligand-induced conformational changes are essentially localized in the heme pocket area and effect proximal side residues more than the distal side ones. Some notable for the functional mechanism. [ABSTRACT FROM AUTHOR]

Type VIII collagen molecules, expressed by cornea! and vascular endothelial cells, appear to be heterotrimers composed of two genetically distinct polypeptides, α1 (VIII) and α2( VIII), in the ratio of 2:1. Characterization of the rabbit α1(VIII) gene has demonstrated that it consists of only four exons, one of which is large and encodes the entire triple-helical and carboxyl non-triple-helical domains. A similar exon organization has been found for the chicken α1(X) and the mouse and human α2(VIII) collagen genes. The genes encoding α1(VIII), α2(VIII), and α1(X) collagen chains are therefore homologous members of a unique class of genes within the collagen superfamily. In the present paper we describe for the first time the primary structure of the human α1(VIII) collagen chain, based on isolation and sequencing of a genomic DNA fragment. The results indicate that the human α1(VIII) chain has the same domain structure as the rabbit protein. We also demonstrate that the gene is localized on the long arm of the human chromosome 3. The availability of genomic DNA encoding the human α1(VIII) collagen chain and information about its chromosomal location should make it possible to examine whether hereditary diseases are linked to abnormalities in the structure or expression of the α1(VIII) gene. [ABSTRACT FROM AUTHOR]

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

A 1.4-kb Pstl - HpaI DNA fragment carrying the Kiebsiella pneumoniae nifM gene has been sequenced; nifM has been shown to encode a 30.64Da polypeptide. Two other open-reading frames were identified upstream of nifM. The one immediately upstream of nziM encodes a 16.6-kDa polypeptide which has been identified by in vitro transcription/translation in an Escherichia coli 30000 × g supernatant system; we propose to designate this gene njfM. The sequence of the second open reading frame is incomplete but it does not correspond to nifV, the gene previously thought to be immediately upstream of nifM, and may therefore identify another new nif gene. Both nifM and nifM have functional n?f promoters with the characteristic -24, .42 consensus sequence, we find no evidence for a nifM upstream activator sequence. The role of nifZ in nitrogenase biosynthesis is unknown but its identification calls into question previous assertions that only nifM and nifH are required for the synthesis of nitrogenase Fe protein. [ABSTRACT FROM AUTHOR]

The interspecific hybrid Arbacia lixula ... × Paracentrotus lividus ... which stops developing at the onset of gastrulation, was previously found to contain much more DNA of maternal origin (P. lividus) than DNA of paternal origin. In spite of this, the RNA synthesized by the blocked gastrula hybridizes better with DNA of paternal type than with DNA of maternal type. An explanation of thru observation was sought in the present paper by means of saturation and competition experiments. The higher affinity of RNA from the hybrid gastrula for paternal DNA could be due to the fact that RNA of paternal type is complementary to a longer portion of the corresponding DNA than RNA of maternal type. This explanation was not satisfactory since the number of genes of paternal type which are active in the hybrid is not very different from the number of genes of maternal type. In normal embryos of A. lixula and P. lividus, RNA from unfertilized eggs and from plutei was found to be less competitive against labeled RNA from gastrulae than gastrula RNA itself. Transcription of the genome is therefore partially stage-specific. This stage-specificity is maintained in the hybrid as far as transcription of the maternal genome is concerned, but is lost as far as transcription of the paternal genome is concerned. The hybrid gastrula was shown to synthesize more RNA molecules of A. lixula type than does the normal gastrula of this species, whereas RNA of P. lividus type is produced in equal amounts m normal and in hybrid embryos. These observations explain the hybridization properties of the RNA synthesized by the blocked gastrula. The results suggest that the genes of paternal type that are very active in the hybrid are those genes which m normal embryos are transcribed at all stages of development and probably also in the adult. This class of genes is much more active in a foreign cytoplasm than in the cytoplasm of its own species. On the other hand, the genes whose activity is restricted to the gastrula stage m normal embryos are not active in the hybrid. This class of genes does not seem to function in a foreign cytoplasm. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the beta-xylosidase gene (xynB) of Bacillus pumilus IPO and its flanking regions was established. A 1617-bp open reading frame for beta-xylosidase, a homodimer enzyme, was observed. The amino acid sequence of the N-terminal region and the molecular mass 62607 Da) of the beta-xylosidase subunit, deduced from the DNA sequence, agreed with the result obtained with the purified enzyme. The Shine-Dalgarno sequence was found 8 bp upstream of the initiation codon, ATG. The xylanase gene (xynA) of the same strain was 4.6 kbp downstream of the 3' end of xynB, and its DNA sequence was reported in our previous paper [Fukusaki, E., Panbangred, W., Shinmyo, A. & Okada, H. (1984) FEBS Lett. 171, 197-201]. The results of the Northern hybridization suggested that the mRNA of xynA and xynB were produced separately. The 5' and 3' ends of the xynA and xynB gene were mapped with nuclease S1. The '-10' regions for promoter sequences of both genes were similar to the consensus sequence for B. subtilis RNA polymerases, the '-35' regions were different from all the known promoters for B. subtilis RNA polymerases.

The complete nucleotide sequence of the 16-S RNA from Escherichia coli has been determined using rapid RNA-sequencing gel methods. The experimental data are fully described in this paper. The specificities of the ribonucleases, especially the ribonuclease PhyI are discussed and the consequences of the persistence of stable secondary structure are considered. The proposed sequence contains 1541 nucleotides and agrees completely with the DNA sequence of the rrnB cistron deduced by Brosius, J., Palmer, M.L., Kennedy, P.J., and Noller, H.F. [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 4801-4805]. But there are several cistron heterogeneities of which we described 16 single-base heterogeneities, 7 of the deletion/insertion type and 9 of the transition or transversion type. Our observations suggest the existence, among the 7 ribosome RNA cistrons, of one or two mutated ones. The respective advantages and disadvantages of both RNA and DNA sequencing methods are discussed.

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.

An 11,450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high-copy-number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DD-peptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine beta-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coli ampC beta-lactamase (class C). Since the Streptomyces R61 DD-peptidase and beta-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the beta-lactamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the beta-lactamases of classes A and C are related in an evolutionary sense.

The incubation of 25-S tobacco mosaic virus (TMV) protein with a mixture of RNA fragments produced by partial T1 RNase digestion of TMV RNA results in the encapsidation of only a few species of RNA. In addition to the most predominant species, fragment 1, whose sequence has been described in the prededing paper, two other species, fragment 41 and fragment 21 are coated by the protein. These two RNA fragments were purified by polyacrylamide gel electrophoresis and subjected to total digestion with pancreatic and T1 RNase. The oligonucleotides were separated by paper electrophoresis and characterized insofar as possible by digestion with the complementary ribonuclease. From the amino acid coding capacity of the oligonucleotides liberated from fragments 41 and 21 by T1 RNase digestion, it appears that these two fragments, like fragment 1, are derived from the coat protein cistron. They are situated immediately prior to fragment 1 and, together with this fragment, consitute a continuous stretch of 232 nucleotides of the cistron which codes for animo acids 53 to 130 of the coat protein. The order of the fragments in the sequence is 21-41-1. A possible model for the secondary structure of this portion of the sequence is proposed.

The article presents information on several research papers related to the biochemistry and medical education. Some of the topics discussed are: Problem-based learning in medical course, Genes involved in oxytocin-induced cardiomyogenesis, Polyamine oxidase activity in malignant ascites pancreatic origin, Oxidative stress of the red blood cells treated with tert-butyl hydroperoxide, Detection of circulating breast cancer cells in peripheral blood by a two-marker RT-PCR assay, Expression of CYP 1A1, CYP1B1 and CYP3A4 genes in normal endometrium and endometrial cancer cells.

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCI profiles and d number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences. [ABSTRACT FROM AUTHOR]

The structure of minichromosomes from an SV40 recombinant carrying a functional mouse α-globulin gene in its late coding region was studied by DNase I treatment of isolated nuclei from infected cells. Ten prominent DNase- l-hypersensitive sites were detected in the inserted globin fragment, six in the region upstream from the transcription initiation site and four within the gene itself. Corresponding sites were not seen in naked DNA, nor in minichrosomes without this insertion. However this hypersensitivity pattern differs from that observed for mouse α- globin in Friend erythroleukemia cells before or aftcr induction. The recombinant minichromosome also displayed eight hypersensitive sites, indistinguishable from those seen in unsubstituted SV40, in the viral sequences containing the origin of replication and the regulatory signals for early and late viral transcription. The cleavage sites in SV40 occur predominently in the origin-promotor-enhancer region, whereas those in the globin fragment were more dispersed and less pronounced. This suggests that the specific local opening of this region in the viral chromatin is not due solely to the presence of active promoters, but is associated with the function of the enhancer element. [ABSTRACT FROM AUTHOR]

Mesenchyme blastula sea-urchin embryos were heat-shocked at 31 °C and pulse-labelled with [3H]uridine. The nuclear RNA was fractionated on glyoxal/agarose gels and each RNA fraction hybridized with cloned early blastula histone DNA. The results showed that after heat shock there is an accumulation of histone RNA molecules larger than the messenger and a decrease, with respect to the control, of 9-S histone RNA. Chasing of the heat shock RNA by incubation of the embryos with unlabelled uridine and cytidine restores the radioactivity, as well as the hybridization profiles, of control embryos. Furthermore saturation curves obtained by hybridizing histone DNA with labelled 9-S RNA, showed an absence of the histone messenger in the cytoplasm of heat-shocked embryos, whereas it is present in both control and chased embryos. These results are consistent with the hypothesis that the accumulated histone RNA in heat-shocked embryos is the precursor of mature messengers. [ABSTRACT FROM AUTHOR]

We have studied RNA transcripts of the defective parvovirus, adeno-associated virus (AAV) present in poly-(A)rich and poly-(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly-(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 × 103, 3.3 × 103 and 2.3 × 103 bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 × 103, 3.6 × 103 and 2.6 × 103 bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)-free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 × 103 bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 × 103 and 2.8 × 103 bases. The 4.2 × 103 and 3.6 × 103-base unspliced RNAs are more abundant than the coterminal 3.9 × 103 and 3.3 × 103-base spliced RNAs whereas the 2.3 × 103-base spliced RNA is much more abundant than the 2.6 × 103-base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post-transcriptional events of splicing or message stability. We also analysed the effects of AAV defective-interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10-fold by defective-interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective-interfering genomes occurs mostly at the level of DNA replication rather than transcription. [ABSTRACT FROM AUTHOR]

The substrate specificity of 49[SUP+]-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (> 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49[SUP+]-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49[SUP+]-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA. [ABSTRACT FROM AUTHOR]

1. Comparison of DNA-DNA filter hybridization using either denaturated duplex DNA or artificial mixtures of the two complementary strands of T7 DNA shows that the competing renaturation reaction has two effects: (a) sequestering of 20-30 % of the input DNA as duplex, unavailable for hybridization, and (b) hybridization of partially renatured DNA as a concatenane to the filter-bound DNA. 2. Experiments using artificial mixtures of 32P-labelled and ³H-labelled L and H strands of T7 show that I0-20% of the total hybrid bound results from concatenation. With a 1: 1 input ratio of complementary strands in solution, 10% of the input bound to filter DNA of a single strand (e.g. L) is of the homologous strand (i.e. L). This effect is relatively unaffected by the concentration of DNA in solution. 3. Variation of the L:H ratio in solution shows that the strand in excess hybridizes and renatures more efficiently. This effect gives an underestimation of the amount of the minor strand present in solution. The numerical values obtained here are likely to be strongly dependent on the complexity of the DNA and the precise hybridization conditions. 4. The implication of the results for homology experiments and determination of strand specificity in transcription is discussed. [ABSTRACT FROM AUTHOR]

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD-peptidase was fragmented by (a) cyanogen bromide cleavage of the carboxymethylated protein, (b) trypsin digestion of the carboxymethylated protein and (c) trypsin digestion of the protein treated with β-iodopenicillinate and endoxo-Δ14-tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, β-iodopenicillanate reacted selectively with the active site of the DD-peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active-site peptide. [ABSTRACT FROM AUTHOR]

The tyrosine tRNAs specified by the E. coli amber suppressor gene suIII+ and the wild type non-suppressing allele suCH were selectively labelled with 32P in E. coli infected with transducing phage φ80 carrying either of these genes. The determination of the nucleotide sequence of the suIII+ tyrosine tRNA is described. This tRNA has the anticodon CUA. The suIII- tRNA differs from suIII+ tRNA by a single nucleotide and has the anticodon GUA where G is a modified guanylic acid residue. E. coli su- cells contain two tyrosine tRNAs (I and II). The determination of the nucleotide sequence of these is described. Species I is identical to suIII- tRNA while species II differs by two nucleotides in the variable loop region. [ABSTRACT FROM AUTHOR]

Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism. TheINO1gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in thesec14Δ cki1Δ strain (SEC14encodes the major yeast phosphatidylinositol transfer protein;CKI1encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrantINO1gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of theINO1gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis[Henry, S.A.&Patton-Vogt, J.L. (1998)Prog. Nucleic Acid Res. Mol. Biol.61, 133–179]. [ABSTRACT FROM AUTHOR]

HIC1(hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified bothin vivoandin vitrowith O-linkedN-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of[3H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites forO-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown thatO-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that theO-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399–714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised againstO-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins areO-GlcNAc modified in their C-terminal tail (residues 670–711) and not in the DNA-binding domain, as for the full-length proteins. Thus, theO-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress. [ABSTRACT FROM AUTHOR]

A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library. The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5). In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX4CX2H consensus sequence. Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization. The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues. These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice α-amylase gene. [ABSTRACT FROM AUTHOR]

The properties of the Δ6 desaturase/acetylenase from the moss Ceratodon purpureus and the Δ12 acetylenase from the dicot Crepis alpina were studied by expressing the encoding genes in Arabidopsis thaliana and Saccharomyces cerevisiae. The acetylenase from C. alpinaΔ12 desaturated both oleate and linoleate with about equal efficiency. The desaturation of oleate gave rise to 9( Z),12( E)- and 9( Z),12( Z)-octadecadienoates in a ratio of approximately 3 : 1. Experiments using stereospecifically deuterated oleates showed that the pro-R hydrogen atoms were removed from C-12 and C-13 in the introduction of the 12( Z) double bond, whereas the pro-R and pro-S hydrogen atoms were removed from these carbons during the formation of the 12( E) double bond. The results suggested that the Δ12 acetylenase could accommodate oleate having either a cisoid or transoid conformation of the C12-C13 single bond, and that these conformers served as precursors of the 12( Z) and 12( E) double bonds, respectively. However, only the 9( Z),12( Z)-octadecadienoate isomer could be further desaturated to 9( Z)-octadecen-12-ynoate (crepenynate) by the enzyme. The evolutionarily closely related Δ12 epoxygenase from Crepis palaestina had only weak desaturase activity but could also produce 9( Z),12( E)-octadecadienoate from oleate. The Δ6 acetylenase/desaturase from C. purpureus, on the other hand, produced only the 6( Z) isomers using C16 and C18 acyl groups possessing a Δ9 double bond as substrates. The Δ6 double bond was efficiently further converted to an acetylenic bond by a second round of desaturation but only if the acyl substrate had a Δ12 double bond and that this was in the Z configuration. [ABSTRACT FROM AUTHOR]

Little is known about the functions of DNA polymerase λ (Pol λ) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol λ may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol λ in a higher plant, rice ( Oryza sativa L. cv. Nipponbare) ( OsPol λ). OsPol λ had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol λ. Northern blot analysis showed that the level of OsPol λ expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol λ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase. [ABSTRACT FROM AUTHOR]

We describe a strategy for systematic amplification of chitin synthase genes ( chs) in the filamentous ascomycetes plant-pathogen Botrytis cinerea using PCR with multiple degenerate primers designed on specific and conserved sequence motifs. Eight distinct chs genes were isolated, named Bcchs I, II, IIIa, IIIb, IV, V, VI and VII. They probably constitute the entire chs multigenic family of this fungus, as revealed by careful analysis of six euascomycetes genomes. Bcchs I, IIIa, IIIb, IV and VI genes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis (HCA) to localize putative residues critical for CHS activity. HCA also enabled us to highlight three different transmembrane topologies of the CHS membranous isoenzymes. We found that the N-terminal region of the BcCHSI isoenzyme, and its orthologues in other euascomycetes, probably contain folded peptide motifs with conserved tyrosine residues. Their putative role is discussed. The BcCHSVII isoenzyme appeared to belong to a new class of CHS orthologues that was demonstrated by phylogenetic study to branch apart from division 1 and 2 of CHS. [ABSTRACT FROM AUTHOR]

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (β2), the recombinant enzyme was a hetero-octameric (β4γ4), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U·mg−1. Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system. [ABSTRACT FROM AUTHOR]

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. Two types of Hdr have been identified and characterized from distantly related methanogens. Here we show that the sulfate-reducing archaeon Archaeoglobus profundus cultivated on H2/sulfate forms enzymes related to both types of Hdr. From the membrane fraction of A. profundus, a two-subunit enzyme (HmeCD) composed of a b-type cytochrome and a hydrophilic iron–sulfur protein was isolated. The amino-terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A. fulgidus. HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp. From the soluble fraction of A. profundus a six-subunit enzyme complex (Mvh:Hdl) containing Ni, iron–sulfur clusters and FAD was isolated. Via amino-terminal sequencing, the encoding genes were identified in the genome of the closely related species A. fulgidus in which these genes are clustered. They encode a three-subunit [NiFe] hydrogenase with high sequence identity to the F420-nonreducing hydrogenase from Methanothermobacter spp. while the remaining three polypeptides are related to the three-subunit heterodisulfide reductase from Methanothermobacter spp. The oxidized enzyme exhibited an unusual EPR spectrum with gxyz = 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr. Upon reduction with H2 this signal was no longer detectable. [ABSTRACT FROM AUTHOR]

The brain fatty acid-binding protein (B-FABP) is involved in brain development and adult neurogenesis. We have determined the sequence of the gene encoding the B-FABP in zebrafish. The zebrafish B-FABP gene spans 2370 bp and contains four exons interrupted by three introns. The coding sequence of zebrafish B-FABP gene is identical to its cDNA sequence and the coding capacity of each exon is the same as that for the human and mouse B-FABP genes. A 1249 bp sequence 5′ upstream of exon 1 of the zebrafish B-FABP gene was cloned and sequenced. Several brain development/growth-associated transcription factor binding elements, including POU-domain binding elements and the proposed lipogenic-associated transcription factor NF-Y elements, were found within the 5′ region of the B-FABP gene. RT-PCR analysis using mRNA extracted from different tissues of adult zebrafish demonstrated that the zebrafish B-FABP mRNA was predominant in brain with lower levels in liver, testis and intestine, but not in ovary, skin, heart, kidney and muscle. Quantitative RT-PCR revealed a similar tissue-specific distribution for zebrafish B-FABP mRNA except that very low levels of B-FABP mRNA, normalized to β-actin mRNA, were detected in the heart and muscle RNA, but not in liver RNA. Zebrafish B-FABP mRNA was detected by RT-PCR in embryos beyond 12 h postfertilization, suggesting a correlation of zebrafish B-FABP mRNA expression with early brain development. Radiation hybrid mapping assigned the zebrafish B-FABP gene to linkage group 17. Conserved syntenies of the zebrafish B-FABP gene and the human and mouse orthologous B-FABP genes were observed by comparative genomic analysis. [ABSTRACT FROM AUTHOR]

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [ABSTRACT FROM AUTHOR]