New regulatory mutations affecting the expression of the threonine operon in Escherichia coli K-12

Summary The promoter of the threonine operon was joined to the structural genes of the lac operon in Escherichia coli K12. The synthesis of -galactosidase was thus repressed by threonine plus isoleucine in the fusion strains. To isolate mutations which affect the expression of the threonine operon, alterations in the level of expression of the lacZ gene were selected. A new type of regulatory mutation was discovered.This paper is part of a thesis presented by Isabelle Saint-Girons in partial fulfillment of the requirements for the Dr. Sc. degree from the University of Paris     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin , was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M _r 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin . It is concluded that conglutin is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.     

Amyloid beta-peptide promotes permeability transition pore in brain mitochondria

In this work the effect of the neurotoxic amino acid sequence, A_25–35, on brain mitochondrial permeability transition pore (PTP) was studied. For the purpose, the mitochondrial transmembrane potential (m), mitochondrial respiration and the calcium fluxes were examined. It was observed that A_25–35, in the presence of Ca²⁺, decreased the m, the capacity of brain mitochondria to accumulate calcium and led to a complete uncoupling of the respiration. However, the reverse sequence of the peptide A_25–35 (A_35–25) did not promote the PTP. The alterations promoted by A_35–25 and/or Ca²⁺ could be reversed when Ca²⁺ was removed by EGTA or when ADP plus oligomycin were present. The pre-treatment with CsA or ADP plus oligomycin prevented the m drop and preserved the capacity of mitochondria to accumulate Ca²⁺. These results suggest that A_25–35 can promote the PTP induced by Ca²⁺.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

The molecular defect in propionic acidemia: Exon skipping caused by an 8-bp deletion from an intron in the PCCB allele

Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. To investigate the genetic basis of propionic acidemia, we isolated a cDNA encoding the precursor of the subunit of human PCC ( PCC). The cloned cDNA sequence was 1,832 bp long and the open reading frame of 1,617 nucleotides encoded a polypeptide of 539 amino acids with a molecular mass of 58,202 Da. The human PCC sequence shared a high degree of homology (91%) with the full-length cDNA of rat PCC at the amino acid level; there were only 47 differences among 539 amino acid residues compared. Polymerase chain reaction amplification and sequencing of cDNA from a subunit-deficient Japanese patient revealed a deletion of 101 nucleotides consisting of one exon from mature mRNA. This deletion resulted in a frameshift and a stop codon in the new frame. Analysis of the genomic DNA revealed a homozygous 8-bp deletion from bp3 to bp10 of the intron just downstream of the deleted exon. This deletion disrupted the consensus 5 splice signal and led to exon skipping.     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

The activity of nitrate reductase and the pool sizes of some amino acids and some sugars in water-stressed maize leaves

The activity of nitrate reductase and the pool sizes of some amino acids and some sugars were measured in relation to the leaf water potential () of maize leaves. The activity of nitrate reductase was severely inhibited in water-stressed maize leaves. This was not due to substrate shortage or the presence of an inhibitor at reduced leaf water potential. While the typical proteinogenic amino acids valine, tyrosine, leucine and isoleucine were almost undetectable in the leaves of the control plants, their concentrations markedly increased with declining , thus indicating protein degradation. The concentrations of serine, glycine and glutamate increased upon water stress, their total amount in severely stressed leaves ranging 5- to 6-fold higher than the total amount of valine, tyrosine, leucine and isoleucine at this stage of water deficit. The pool sizes of glucose, fructose and sucrose decreased in relation to decreasing . The total amount of organic solutes remained almost constant at least up to a of approx.—1.0 MPa and then dropped to about 50% when reached –1.25 MPa.Abbreviations PCR photosynthetic carbon reduction cycle - PCO photosynthetic carbon oxidation cycle - PAR photosynthetically active radiation     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Mussel glue fromMytilus californianus Conrad: a comparative study

Summary Marine mussels secrete a byssus in order to attach to solid surfaces in the sea. The polyphenolic protein is the glue in the adhesive plaques of the byssus. InMytilus californianus, the polyphenolic protein has an apparent molecular weight of 85,000±5,000 and is rich in the amino acids lysine, 3,4-dihydroxyphenylalanine, serine, threonine, and hydroxyproline. In composition it resembles the polyphenolic protein ofM. edulis (M _r=125,000), although theM. edulis protein contains significantly less isoleucine and more alanine. Tryptic digestion ofM. californianus polyphenolic protein revealed two types of repeating decapeptides (1) (Ser/Thr)-Thr-(Tyr/Dopa)-Hyp-Hyp-Thr-Dopa-Lys-Hyp-Lys and (2) Ile-(Thr/Ser)-(Tyr/Dopa)-Hyp-Hyp-Thr-Dopa-Lys-Hyp-Lys. Residues 2 to 8 are identical with residues 4–10 inM. edulis decapeptides.Abbreviation Dopa 3,4hydroxyphenylalanine     

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor.     

Distinct affinity and effector residues in the binding site for a regulatory ligand

A hypothesis concerning two distinct classes of amino acid residues in some regulatory binding sites is proposed. The affinity residues are those that are unable to transduce the ligand information signal but are responsible for overcoming the barrier for the attachment of a ligand to its binding site while the effector residues transfer the binding signal to the other functional part of the protein, which then undergoes a non-equilibrium energetic cycle induced by interaction with the ligand.As an example, the purine nucleotide inhibition of H⁺ transport through the uncoupling protein of brown adipose tissue mitochondria is discussed; there is a concentration range in which the nucleotide is bound but does not inhibit H⁺ transport. This is interpreted in terms of inaccessibility of the effector residues inducing H⁺ transport inhibition below a certain threshold concentration.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

HbF-Lesvos: An HbF variant due to a novel Gγ mutation (:Gγ 75 ATA→ACA) detected in a Greek family

A TC substitution at position 402 of the G globin gene results in an isoleucine to threonine substitution at codon 75 of the G globin chain and the formation of HbF-Lesvos [₂G₂ 75 (E19) IleThr].     

Stimulation of β-carotene synthesis by hydrogen peroxide in Blakeslea trispora

-Carotene synthesis was increased from a negligible amount to 152 mg (g-dry cells)^–1 and H₂O₂ was accumulated up to 16.7 M during 2.5 day-culture of Blakeslea trispora. When cells were cultivated in 250 ml flasks containing various volumes (25–150 ml) of the medium, not only H₂O₂ accumulation but also -carotene synthesis increased as culture volume decreased. Addition of H₂O₂ (10 M) to the 1.5-day old cultures of B. trispora resulted in 46% higher -carotene synthesis than that without addition. All these results indicate that -carotene biosynthesis is stimulated by H₂O₂ in B. trispora.     

Rapid microbial metabolism of non-protein amino acids in the sea

The non-protein amino acids, -alanine and -aminobutyric acid, frequently dominate the amino acid composition of deep-sea sediments. This accumulation is most likely due to the slower decomposition of non-protein amino acids by microorganisms or to the preferential adsorption of non-protein amino acids by clay minerals. We investigated relative rates of heterotrophic uptake of alanine, -ala, and -aba in sea water to see if there were different rates of microbial assimilation and respiration between these protein and non-protein amino acids. Heterotrophic uptake was rapid for all three amino acids with turnover times of hours in productive coastal waters and days in more oligotrophic open-ocean waters. Uptake of the non-protein amino acids was significantly slower than uptake of alanine, particularly in anoxic waters. However, the difference in uptake rates is probably not great enough to cause significant preferential accumulation of non-protein amino acids.     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.