Alcohol production by selected yeast strains in lactase-hydrolyzed acid whey

Ethanol production by Kluyveromyces fragilis and Saccharomyces cerevisiae was studied using cottage cheese whey in which 80 to 90% of the lactose present had been prehydrolyzed to glucose and galactose. Complete fermentation of the sugar by K. fragilis required 120 hr at 30°C in lactase-hydrolyzed whey compared to 72 hr in nonhydrolyzed whey. This effect was due to a diauxic fermentation pattern in lactase-hydrolyzed whey with glucose being fermented before galactose. Ethanol yields of about 2% were obtained in both types of whey when K. fragilis was the organism used for fermentation. Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K. fragilis, but galactose was fermented only when S. cerevisiae was pregrown on galactose. Slightly lower alcohol yields were obtained with S. cerevisiae, owing to the presence of some lactose in the whey which was not fermented by this organism. Although prehydrolysis of lactose in whey and whey fractions is advantageous in that microbial species unable to ferment lactose may be utilized, diauxie and galactose utilization problems must be considered.     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Electron microscope heteroduplex mapping of naphthalene oxidation genes on the NAH7 and SAL1 plasmids

Introduction of the transposon Tn5 to serve as a marker allows electron microscope heteroduplex mapping of the naphthalene oxidation genes on the approximately 83-kb NAH7 and the related approximately 85-kb SAL1 plasmids. The electron microscope-mapped gene positions on the NAH7 plasmid are in close agreement with those mapped previously by restriction digestion. The SAL1 plasmid can be considered as a mutant NAH7 plasmid which fails to direct the conversion of naphthalene to salicylate because of a mutational block but retains intact coding sequences for salicylate oxidation. Analysis of heteroduplex molecules formed between the SAL1 and NAH7::Tn5 EcoRI fragments and the known NAH7/SAL1 homology strongly suggest that the SAL1 DNA is completely homologous to NAH7 DNA except that a approximately 2.5-kb DNA segment constituting most of the nahA gene is replaced by approximately 4.6-kb nonhomologous DNA.     

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5 and 3 flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallization and preliminary X-ray investigation of a ubiquitin carrier protein (E2) from Arabidopsis thaliana.

Crystals of a recombinant ubiquitin carrier protein from Arabidopsis thaliana have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 41.8(1) A, b = 44.9(1) A and c = 83.2(1) A. The crystals are quite stable to X-rays and diffract beyond 2.1 A resolution. There is one molecule in the asymmetric unit.     

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.