Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

A comparison of sweating responses during exercise and recovery in terms of sweating rate and body temperature

Based on the hypothesis that the relation between sweating rate and body temperature should be different during exercise and rest after exercise, we compared the sweating response during exercise and recovery at a similar body temperature. Healthy male subjects performed submaximal exercise (Experiment 1) and maximal exercise (Experiment 2) in a room at 27° C and 35% relative humidity. During exercise and recovery of 20 min after exercise, esophageal temperature (Tes), mean skin temperature, mean body temperature ( ), chest sweating rate ( ), and the frequency of sweat expulsion (F _SW) were measured. In both experiments, andF _SW were clearly higher during exercise than recovery at a similar body temperature (Tes, ). was similar during exercise and recovery, or a little less during the former, at a similarF _SW. It is concluded that the sweating rate during exercise is greater than that during recovery at the same body temperature, due to greater central sudomotor activity during exercise. The difference between the two values is thought to be related to non-thermal factors and the rate of change in mean skin temperature.     

Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium.

Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.     

Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium.

1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.     

Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.