Purification and partial characterization of a flocculin from brewer's yeast.

Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin. The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000. The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1. The flocculin was isolated from flocculent S. cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells. The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).     

Insulin-like activity of chromium-binding fractions from brewer's yeast

51CrCl3 was added to the incubation medium of Saccharomyces cerevisiae for up to 48 hr. After repeated freezing and thawing, lysing in 9 M urea with 1% NP-40 detergent, and dialysis against water, the lower molecular weight (Mr less than 3500) dialysate was retained on a SE53 cationic exchange column, eluted with 0.25 M NH4OH and fractionated on a Bio-gel P-2 column. The insulin-like biological activity of the fractions was measured by the 14C-glucose oxidation in isolated rat adipocytes. The biological activity that was found in two of nine fractions did not correspond to their chromium content. Moreover, identical findings were obtained when chromium was added not to the live yeast but to the yeast extract, which showed that its binding was a chemical process not requiring cellular activity. No fraction demonstrated insulin-potentiating activity on rat adipocytes.     

The nucleotide sequence of asparagine tRNA from brewer's yeast

The nucleotide sequence of asparagine tRNA from brewer's yeast has been determined using postlabeling methods. The primary structure is as follows: pG-A-C-U-C-C-A-U-G-m2G-C-C-A-A-G-D-D-G-G-D-D-A-A-G-G-C-m2 2G- U-G-C-G-A-C-U-G-U-U -t6A-A-psi-C-G-C-A-A-G-A-D-m5C-G-U-G-A-G-T-psi-C-A-m1A-C-C-C-U-C-A-C-U-G -G-G-G- U -C-G-C-C-A. Its anticodon G-U-U can recognize the two codons for asparagine.     

The structure of yeast tRNAAsp. A model for tRNA interacting with messenger RNA

Abstract

The anticodon of yeast tRNA^Asp, GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA^Asp molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA^Phe. In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA^Asp T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA^Phe. This variation is a consequence of the anticodon-anticodon base pairing which rigidities the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA^Asp substantiate such a correlation.     

Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.

Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.     

Crystal structure of yeast tRNAAsp: atomic coordinates

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.     

Recombinant brewer's yeast strains suitable for accelerated brewing.

Four brewer's yeast strains carrying the alpha-ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The alpha-ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae. In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。