DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

Cajaflavanone and cajanone released from Cajanus cajan (L. Millsp.) roots induce nod genes of Bradyrhizobium sp.

A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium sp (F-4) nod DABC-lacZ translation fusion was constructed and used to monitor nod gene expression in response to pigeonpea root exudate. Two nod-inducing compounds were isolated and identified. Spectral analysis using ultraviolet absorption, infrared spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 5,4-dihydroxy-6-(3-methyl-2-butenyl)-2, 2-dimethyl pyrano-[5, 6:7, 8]-flavanone (cajaflavanone) and 2,4,5-trihydroxy-5-isopentenyl-6, 7-dimethylchromene iso-flavanone (cajanone). When pEA2-21 was introduced into Rhizobium trifolii and R. meliloti cajanone and cajaflavanone did not induce nod gene indicating that specificity of induction appears to be influenced by the host-strain genome.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Alternative splicing of pre-mRNA encoding the <Emphasis Type="Italic">Musca domestica</Emphasis> latrophilin-like protein: Primary structures of four spliced forms of mRNA and their protein products

An internal DNA fragment (2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5- and 3-ends (5- and 3-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (6000 nt) differ in the structures of both the regions encoding signal peptides and 5-terminal untranslated regions. They encode large proteins (1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane strands followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severely truncated at their C-termini (900 aa). They are composed of the domain with only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 175–185.Original Russian Text Copyright © 2005 by Andreev, Danilevich, Grishin.     

Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

A 6′ gentamicin acetyltransferase gene allows effective selection of tobacco transformants using kanamycin as a substrate

6 gentamicin acetyltransferases detoxify aminoglycoside antibiotics containing a 6 amino group. We tested whether a 6 gentamicin acetyltransferase gene (6 gat) of Shigella sp. is suitable as selectable gene in plant transformation using kanamycin (Km) as a substrate. A comparative transformation experiment using Nicotiana tabacum SR1 protoplasts showed that 6 gat is as effective for selection of transformants as the commonly used neomycin phosphotransferase II (nptII). In stably transformed plants we detected moderate levels of the 6 gat mRNA. An enzymatic assay was developed with which the acetyltransferase activity of the protein is easily demonstrated.     

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3,4-diethoxyphenyl)-1,3(dihydroxy)-2-(4'-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial , bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the , bond of 1-(3-4-diethoxyphenyl)-1-(hydroxy)-2-(4'-methoxyphenyl)ethane (XI) also occurred, indicating that a hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2,2(dihydroxy)-2-(4'-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the , bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MS mass spectrometry     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Relative levels of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in some N2 fixing bacteria during derepression and repression of nitrogenase

Addition of ammonium to N₂ fixing cultures of Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum rapidly reduced the intracellular levels of guanosine 5-diphosphate 3-diphosphate (ppGpp) by 70–90%. This change might reflect a regulatory role of ppGpp in nitrogen metabolism.Abbreviations ppGpp guanosine 5-diphosphate 3-diphosphate     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

Summary Ten hairy-cell leukemia patients were treated with interferon (IFN-) at a dose rate of 2 × 10⁶ IU/m² × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, 2-microglobulin, (2–5)oligoadenylate [(2–5)A _n ] levels, intracellular (2–5)A _n values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2–5)A _n levels during both induction and maintenance, whereas 2-microglobulin levels rose only during induction. Rises in intracellular (2–5)A _n were documented mainly during induction, but they were not significantly higher than pretherapy values. IFN provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFN dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFN production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Variants within the 5′-flanking regions of bovine milk protein genes: I. κ-casein-encoding gene

In order to identify DNA variants within the 5-flanking region of the bovine -casein (Cn)-encoding gene, this area of the gene from 13 cows belonging to seven breeds (Holstein Friesian, Brown Swiss, German Simmental, Jersey, Galloway, Scottish Highland and Ceylon Dwarf Zebu) was analysed. For each individual, about 1 kb of the 5-flanking region including exon I was amplified by polymerase chain reaction (PCR). The biotinylated PCR product was immobilized on magnetic beads followed by direct bidirectional sequencing using an automated DNA sequencer. Fifteen DNA variants were identified, some of which are located within potential regulatory sites and possibly involved in the expression of the -casein encoding gene.Abbreviations AP2 activator protein 2 - bp base pair(s) - GRE/RC glucocorticoid response element/reverse complement - HNF3 hepatocyte nuclear factor 3 Cn -casein - MGF mammary gland-specific nuclear factor - nt nucleotid(s), OCT1 octamer-binding site 1 - PA polyacrylamide - PCR polymerase chain reaction - PMF pregnancy-specific mammary nuclear factor - kb kilobase(s) or 1000 bp     

Characterization and localization of fusicoccin-binding sites in leaf tissues of Vicia faba L. probed with a novel radioligand

Tritiated 9-nor-fusicoccin-8-alcohol provides a highly bioactive radioligand of high specific activity which is easily prepared by oxidation of fusicoccin and subsequent reduction with tritiated sodium borohydride. Using this radioligand, we have identified and characterized a selective binding site for fusicoccin (K_a for [³H]-9-nor-fusicoccin-8-alcohol=0.20·10⁹ M^-1; K_a, apparent for fusicoccin=0.21·10⁹ M^-1) located at the plasmalemma of Vicia faba leaf tissue. The site is thermolabile, readily degraded by trypsin and located at the apoplastic face of the plasmalemma based on results obtained using right-side-out plasmalemma vesicles prepared by aqueous two-phase partitioning and macromolecular fusicoccin-derivatives. The binding-protein is present in guard cells of Vicia faba, as shown by the use of purified guard-cell protoplasts.Abbreviations BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FC fusicoccin - FCol 9-nor-fusicoccin-8-alcohol - Mes 2(N-morpholino)ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol