Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 % of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.     

Wiskott–Aldrich syndrome: a gene,a multifunctional protein and the beginnings of an explanation

Patients with Wiskott–Aldrich syndrome show various defects in the normal function of platelets and lymphocytes. The recent identification of the gene responsible for this syndrome has led to a surge of studies aimed at solving the puzzle posed by the varied phenotype observed in this disease. It is now known that WASP, the protein product of this gene, can interact with a large number of other proteins known to be involved in the regulation of signal transduction and cytoskeletal organization. Thus, WASP appears to integrate these two basic and fundamental cellular mechanisms. Several groups are now focusing on understanding the function of WASP in detail, and translating this new knowledge into improved therapies.     

Interaction of chlorophyll a′ with the 65 kDa subunit protein of photosystem I reaction center

The 65 kDa polypeptide subunit depleted of P700 was prepared from a photosystem I reaction center preparation and mixed with chlorophyll a′ (C-10 epimer of chlorophyll a) to yield a complex exhibiting a tripleheaded spectrum with absorbance maxima at 673, 692 and 707 nm. The difference spectra (oxidized-minus-untreated and light-minus-dark) had a major trough at 707 nm and minor ones at 690 and 430 nm. The overall shape of the spectra resembled well that of P700 with a small red shift. A rapidly decaying flash-induced absorbance change was observed at 430 nm with a half decay time of less than 500 μs in a preparation supplemented with an electron donor system.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Characterization of a polymorphism in the 3′ part of the chicken vitellogenin gene

Summary An allele giving rise to a polymorphism within the 3 part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.     

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [¹⁵N-¹H]- and [¹³C-¹H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan ¹⁵N backbone positions and also resolved signals for ¹⁵N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [¹³C-¹H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsare"pathogenesis-relatedproteins"includingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

Attenuated familial adenomatous polyposis due to a mutation in the 3′ part of the APC gene. A clue for understanding the function of the APC protein

The identification of germline mutations in a large number of clinically well-characterised patients with familial adenomatous polyposis (FAP) has allowed the unravelling of several genotype-phenotype relationships that can now be interpreted in the light of the structure and functional domains of the adenomatous polyposis coli (APC) protein. An attenuated phenotype has been found to be associated with mutations at the 5 end of the gene, while a severe clinical expression was found in patients with the most common mutation at codon 1309. So far, only few mutations in the 3 half of the gene have been published. We report on two families with a rather mild phenotype due to a frameshift mutation at codon 1597. These families may represent a clue for defining a 5 border for the occurrence of a second region of attenuated FAP that is localised in the 3 part of the APC gene. We propose a model to explain the relationship between the severity of the disease and the size of the mutant APC protein.     

Generation and characterization of Brassica rapa ssp. pekinensis – B. oleracea var. capitata monosomic and disomic alien addition lines

Journal of Genetics - Five monosomic alien addition lines (MAALs) of Brassica rapa ssp. pekinensis – B. oleracea var. capitata were obtained by hybridization and backcrossing between B. rapa...     

Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the β′ subunit of RNA polymerase

The sequence of a 2782 bp fragment of the chloroplast genome of Chlorella ellipsoidea has been determined. The region includes the entire gene (rbcL) for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase and a sequence (rpoC-like) similar to part of the gene for the subunit of E. coli RNA polymerase which is oriented in same direction as rbcL. The arrangement is rpoC-like — 446 bp — rbcL. The rbcL gene codes for a polypeptide of 475 amino acids whose sequence shows 88% homology with those of tobacco and spinach, 94% homology with that of Chlamydomonas, and 85% homology with that of Anacystis. The putative rbcL promoter sequence has homology with E. coli promoter sequences and its putative terminator sequence is capable of forming a stem-and-loop structure.     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmST_WT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmST_E271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmST_E271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.     

An evolutionary analytical model of a complementary circular code simulating the protein coding genes, the 5′ and 3′ regions

The self-complementary subset ∪{AAA,TTT} with = {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} of 22 trinucleotides has a preferential occurrence in the frame 0 (reading frame established by the ATG start trinucleotide) of protein (coding) genes of both prokaryotes and eukaryotes. The subsets ∪{CCC} and ∪{GGG} of 21 trinucleotides have a preferential occurrence in the shifted frames 1 and 2 respectively (frame 0 shifted by one and two nucleotides respectively in the 5′-3′ direction). and are complementary to each other. The subset contains the subset which has the rarity property (6 × 10⁻⁸) to be a complementary maximal circular code with two permutated maximal circular codes and in the frames 1 and 2 respectively. is called a C³ code. A quantitative study of these three subsets in the three frames 0, 1, 2 of protein genes, and the 5′ and 3′ regions of eukaryotes, shows that their occurrence frequencies are constant functions of the trinucleotide positions in the sequences. The frequencies of in the frame 0 of protein genes are 49, 28.5 and 22.5% respectively. In contrast, the frequencies of in the 5′ and 3′ regions of eukaryotes, are independent of the frame. Indeed, the frequency of in the three frames of 5′ (respectively 3′) regions is equal to 35.5% (respectively 38%) and is greater than the frequencies and , both equal to 32.25% (respectively 31%) in the three frames. Several frequency asymmetries unexpectedly observed (e.g. the frequency difference between and in the frame 0), are related to a new property of the subset involving substitutions. An evolutionary analytical model at three parameters (p, q, t) based on an independent mixing of the 22 codons (trinucleotides in frame 0) of with equiprobability (1/22) followed by t ≈ 4 substitutions per codon according to the proportions p ≈ 0.1; q ≈ 0.1 and r = 1 − p − q ≈ 0.8 in the three codon sites respectively, retrieves the frequencies of observed in the three frames of protein genes and explains these asymmetries. Furthermore, the same model (0.1, 0.1, t) after t ≈ 22 substitutions per codon, retrieves the statistical properties observed in the three frames of the 5′ and 3′ regions. The complex behaviour of these analytical curves is totally unexpected and a priori difficult to imagine.     

Doxorubicin-induced neurotoxicity is attenuated by a 43-kD protein from the leaves of Cajanus indicus L. via NF-κB and mitochondria dependent pathways

Doxorubicin (Dox) is an effective anthracycline antitumour drug although its clinical efficacy is restricted because of several acute and chronic side effects. It has been suggested that Dox-induced anticancer effect and neurotoxicity do not follow identical mechanism. The present study has been carried out to investigate the neuroprotecive role of a 43-kD protein (Cajanus indicus (CI) protein) against Dox-induced oxidative impairment and brain tissue damage. Administration of Dox (25 mg/kg body weight) increased reactive oxygen species (ROS) production, altered neuro antioxidant status, activities of brain specific coenzymes (like acetyl coenzyme, monoamine oxidase, etc.), ATPases (like Na(+)/K(+), Ca(2+), etc.) and brain biogenic amines levels. Signal transduction studies showed that Dox markedly decreased mitochondrial membrane potential, disturbed Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased levels of Apaf1, caspase-9/3, cleaved PARP protein and ultimately led to apoptotic cell death. In addition, Dox markedly increased nuclear factor kappa B (NF-κB) nuclear translocation in association with IKKα/β phosphorylation and IκBα degradation. Post-treatment with CI protein (3 mg/kg body weight, once daily for next 4 days), however, reduced Dox-induced oxidative stress, attenuated the nuclear translocation of NF-κB and protected the brain tissue from Dox-induced apoptotic death. Histological studies also support these experimental findings. Results suggest that CI protein might act as a beneficial agent against Dox-induced neuronal dysfunctions.     

(CGG) trinucleotide repeat polymorphism in the 5′ region of the HHR6B gene: the human homolog of the yeast DNA repair gene RAD6

The polymerase chain reaction was used to detect polymorphisms in a (CGG) trinucleotide repeat sequence in the 5 region of the human HHR6B DNA repair gene on chromosome 5q23-31