Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Mosquito circadian and circa-bi-dian flight rhythms: a two-oscillator model

Summary Circadian rhythmicity was found in the flight activity ofCuliseta incidens recorded in constant darkness for up to 14 weeks. The first nonhuman circa-bi-dian (about-two-day) rhythms were also found (Figs. 6–7). Circadian periods were either stable, remaining <24h (Fig. 1), or labile, with a change from <24h to >24 h (Fig. 2). Inactivity phenomena (day-skipping) were common in the latter group only (Fig. 5). The period at activity onset was much more labile than the period at offset (Fig. 4). The activity patterns of some period-lengthened animals suggested control by two oscillators which could temporarily or permanently uncouple (Figs. 8–9).A pacemaker model consisting of a labile evening (E) oscillator mutually coupled to a stable morning (M) oscillator is the most economical proposal which can account for these results. The view that E and M uncouple and run with different periods can account for many records in which the period was labile. Circa-bi-dian rhythms can be explained by the period of E lengthening to where it synchronizes with M in a 21 mode. Thus, E and M are proposed to behave similarly to the human activity and temperature oscillators. It is speculated that day-skipping might indicate that E oscillates between circadian and circa-bi-dian ranges without overt activity being expressed.Abbreviations LD1212 alternating 12h light, 12h dark - DD constant dark - LL constant light - period of rhythm - _on period at activity onset - _off period at activity offset - activity time - mean activity time     

Possible cases of relaxed selection in civilized populations

Summary Genetic traits which are disadvantageous or deleterious in primitive cultures, of hunters and food gatherers, have very low frequencies in almost all cases. There are a few special exceptions, such as the sickle cell trait. Some deleterious traits under genetic control are rare in primitive populations but have fairly high frequencies in populations with long histories or prehistories of civilization, such as Chinese, Japanese or European populations. The Darwinist might suggest that these traits have less selective disadvantage, that is, lower rates of selective elimination, in civilized habitats; i.e. that the various changes in habitat accompanying civilization have caused the rigor of natural selection to relax, in part or perhaps completely.Several cases are discussed of traits under genetic control or genetic influence which demonstrate this phenomenal contrast, between low frequencies among primitive populations and high frequencies among civilized populations. Population data are presented for 4 such cases: colorblindness, myopia, abnormality of the nasal septum and abnormally small external opening of the tear duct.

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Zusammenfassung Mit einigen bekannten Ausnahmen, z. B. Sichelzellanämie, kommen genetische Merkmale, die bei den Urvölkern von Jägern und Sammlern ungünstig oder sogar schädlich wirkten, mit niedriger Häufigkeit vor. Genetisch bedingte schädliche Merkmale, die selten unter Urvölkern gefunden werden, kommen jedoch häufiger vor bei Bevölkerungen, die eine lange Geschichte bzw. Vorgeschichte der Zivilisation besitzen, wie z. B. in China, Japan oder Europa. Ein Darwinist würde behaupten, daß bei zivilisierten Wohnorten solche Merkmale selektiv weniger nachteilig wirken, d. h., daß die selektive Ausscheidung langsamer vorgeht. Anders gesagt: der mit der Zivilisation verbundene Wechsel des Wohnortes bildet einen Grund für eine Milderung der Strenge der natürlichen Auslese.Es werden einige genetisch bestimmte oder genetisch beeinflußte Merkmale besprochen, die den Unterschied zwischen den Häufigkeiten (niedrig bei Urvölkern, hoch bei zivilisierten Völkern) aufzeigen,. Für 4 solcher Fälle: Farbensinnstörungen, Kurzsichtigkeit, Nasenscheidenwand-Deformitäten und anomale kleine Außenöffnungen der Tränenwege werden die Befunde aus den Bevölkerungen dargestellt.

     

Characterization of Baboon (Papio hamadryas) Milk Proteins

The major proteins of baboon milk were identified as -lactoglobulin (LG), -lactalbumin (LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human LA, lysozyme, and albumin and bovine LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon LG are identical to those of macaque (Macaca fasicularis) LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of LG were elucidated using RT-PCR amplification of poly(A)⁺ mRNA purified from lactating mammary gland. Baboon LG consists of 168 amino acid residues (M_r 20,750) and is the longest LG identified to date. LG and LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4–6, of individual baboon milk samples at varying stages of lactation.     

The single DR β gene of the DRw8 haplotype is closely related to the DR β 3III gene encoding DRw52

In most individuals two HLA-DR genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DR pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DR gene. To determine the relationship of this single gene to the multiple DR genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DR chain locus were included. The DR w8 gene was indistinguishable from the DR III gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.     

Differences in the heat-shock response between thermotolerant and thermosusceptible cultivars of hexaploid wheat

Summary Heat-shock protein (HSP) gene expression in two wheat lines cv Mustang (heat-tolerant) and cv Sturdy (heat-susceptible) were analyzed to determine if wheat genotypes differing in heat tolerance also differ in in-vitro HSP synthesis (translatable HSP mRNAs) and steady-state levels of HSP mRNA. Several sets of mRNA were isolated from seedling leaf tissues which had been heat-stressed at 37 °C for various time intervals. These mRNAs were hybridized with HSP cDNA or genomic DNA probes (HSP17, 26, 70, 98, and ubiquitin). Protein profiles were compared using in-vitro translation and 2-D gels. The Northern slot-blot data from the heat-stress treatment provide evidence that the heat-tolerant cv Mustang synthesized low molecular weight (LMW) HSP mRNA earlier during exposure to heat shock and at a higher level than did the heat-susceptible cv Sturdy. This was especially true for the chloroplast-localized HSP. The protein profiles shown by 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between the two wheat lines, but also some unique HSPs which were only found in the Mustang HSP profiles. The high level of RFLP between the two wheat lines was revealed by Southern blot hybridization utilizing a HSP17 probe. These data provide a molecular basis for further genetic analysis of the role of HSP genes in thermal tolerance in wheat.     

High misses after odd flashes in oxygen evolution in thoroughly dark-adapted thylakoids from pea and Chenopodium album

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, Q_B. It has been generally believed that during the flash-induced transition of each of the S-states (S_n S_n+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, miss being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S₁ state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S₀ S₁, S₁ S₂, S₂ S₃ and S₃ S₀ transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the bicycle model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834–1838), where misses are due to the presence of P⁺ or Q_A ^- among the various equilibrium states of PS II centers.Abbreviations miss parameter - double hit parameter - PS II Photosystem II - Q_A primary one-electron acceptor of PS II, a plastoquinone molecule - Q_B secondary plastoquinone two-electron acceptor of PS II - S-states (S_n, where n=0, 1, 2, 3 or 4) redox states of the oxygen evolving complex     

Histochemical localization of 3 - and 17 -hydroxysteroid dehydrogenases in the male reprodutive tract of the domestic fowl (Gallus domesticus)

Synopsis 3- and 17-hydroxysteroid dehydrogenase activities were studied histochemically in the male reproductive tract of the domestic fowl. 3-hydroxysteroid dehydrogenase was NAD⁺-linked and was capable of metabolizing the three substrates used, namely, pregnenolone, 17-hydroxypregnenolone and dehydroepiandrosterone. 17-hydroxysteroid dehydrogenase oxidized testosterone in the presence of NAD⁺ or NADP⁺. The pattern of distribution of formazan granules was essentially the same with all the substrates used, and they were located in the Leydig cells, seminiferous tubules and the lining epithelia of the entire excurrent duct system of the testis except the rete testis. The activity of both enzymes appeared to be highest in the ductuli efferentes and decreased distally along the tract. The evidence suggests that steroid synthesis may occur in the epithelial lining of the excurrent ducts as well as in the cells of the testis.     

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.     

Modification of nuclear gene expression by inhibition of mitochondrial translation during sporulation in MAT alpha/MATa diploids of Saccharomyces cerevisiae

Summary Sporulation of S. cerevisiae MAT/MATa was accompanied by a novel pattern of protein synthesis as shown by the disappearance of some mitotic polypeptides and by the appearance of a new set of meiotic polypeptides. Inhibition of mitochondrial protein synthesis by erythromycin within the 1st h caused the disappearance of several meiotic polypeptides. These meiotic polypeptides were also sensitive to cycloheximide and were localized in the cytosol, demonstrating that they were not mitochondrial translational products. Since erythromycin affected neither protein synthesis nor sporulation in a mitochondrially inherited er y mutant, we conclude that mitochondrial protein synthesis is needed for the expression of some nuclear genes during sporulation.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Binding of organic solvent molecules influences the P1′−P2′ stereo-and sequence-specificity of α-chymotrypsin in kinetically controlled peptide synthesis

In kinetically controlled peptide synthesis catalyzed by -chymotrypsin (EC 3.4.21.1, CT) the P₁-specificity and stereospecificity was found to increase for hydrophobic and decrease for positively charged nucleophiles in systems with hydrophobic organic solvent molecules. This was observed in the following systems: (i) buffer saturated with organic solvent molecules; (ii) the enzyme adsorbed on a hydrophobic adsorbent (Phenylbutylamine-Eupergit). These results show that organic molecules bound to the enzyme surface perturb the P₁-S₁ interactions and change the catalytic properties of the enzyme.     

Partial sequence of hemoglobin from cobra (Naja naja naja)

Hemoglobin from the cobra snake, Naja naja naja, was isolated and its chains separated on a CM-cellulose column. The separation profile revealed an and two chains having the molar proportions of []₂,[ ₁]₁,[ ₂]₁. The N-terminal amino acid sequence of the intact chains and of the CNBr peptides were carried out. The ₂ chain was found to be heterogeneous comprising a minor component amounting to 11%. This later showed changes at two positions 9 and 14 in the first 30 residues sequenced.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Purification and characterization of alpha-galactosidase from sunflower seeds

From 100 g sunflower seeds, 1.2 mg purified -galactosidase was obtained with an overall yield of 51%. The -galactosidase acted on both terminal -galactosyl residues and side-chain -galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower -galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and -galactosidases from coffee, guar and Mortierella vinacea -galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Inheritance of the dwarf plant type in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of the dwarf plant type was studied in blackgram (V. mungo (L.) Hepper). Type 9 has erect plant type with normal internode length. The mutant line, EMSD has reduced internode length. The F₁, F₂ and F₃ generations of a cross between Type 9 and EMSD and its reciprocal were studied. The extreme dwarf plant type appeared to be governed by a single recessive gene, dw ₁ dw ₁ with no cytoplasmic effect.Part of Ph.D. Thesis submitted by the first author     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.