Chromatographic separation of ribonucleic acids from bacterial and animal cells on MAB columns]

Practicable and efficient method of the chromatographic separation of ribonucleic acids from animal and bacterial cells on columns with MAB-methylated albumin adsorbed on bentonite basis is developed. Distinct separation of transport and ribosomal RNAs was obtained on MAB columns. Ribosomal RNA from bacterial cells was, in its turn, separated in two fractions. Effect of pH, elution rate and temperature on the efficiency of the fractionation is studied. The method proposed is shown to have a number of advantages as compared with well-known method of RNA fractionation on MAK columns. The essence of the method described is irreversible inactivation of RNAse by AMB, which is confirmed in the series of special experiments.     

A method for separation and determination of cytokinin nucleotides from plant tissues

Recent progress in the study of cytokinin metabolism in plants indicates that quantitative analysis of cytokinin nucleotides is essential for elucidation of early steps of the biosynthetic pathway. However, traditional procedures for purification and quantification of cytokinin cannot discriminate the various nucleotides. We describe here a method for separation and determination of cytokinin nucleotides through a series of anion-exchange column chromatography steps followed by liquid chromatography-mass spectrometry. This method enabled us to analyze the amount of each species of cytokinin nucleotide in plant tissues.     

Application of polyethyleneimine cellulose for the class separation of steroidal carboxylic acids from neutral steroids and pigments in urine

Metabolites of corticosteroids that contain the 21-oic acid moiety are found in human urine. The acids from neutral steroids and urinary pigments have been separated by passing the mixture through a column of polyethyleneimine cellulose. The acids adhering to the column are quantitatively eluted with dilute formic acid. The purified preparation is suitable for derivatization and chromatographic analysis.     

Purification, identification and properties of cysteine cathepsins from animal tissues

The paper deals with the modern methods of purification of cathepsins B, H, L, N, S. The ways to control their separation in the course of purification are discussed. The multiplicity of molecular forms of these enzymes is analyzed.     

Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.     

Rapid identification of known and new RNA viruses from animal tissues

Viral surveillance programs or diagnostic labs occasionally obtain infectious samples that fail to be typed by available cell culture, serological, or nucleic acid tests. Five such samples, originating from insect pools, skunk brain, human feces and sewer effluent, collected between 1955 and 1980, resulted in pathology when inoculated into suckling mice. In this study, sequence-independent amplification of partially purified viral nucleic acids and small scale shotgun sequencing was used on mouse brain and muscle tissues. A single viral agent was identified in each sample. For each virus, between 16% to 57% of the viral genome was acquired by sequencing only 42-108 plasmid inserts. Viruses derived from human feces or sewer effluent belonged to the Picornaviridae family and showed between 80% to 91% amino acid identities to known picornaviruses. The complete polyprotein sequence of one virus showed strong similarity to a simian picornavirus sequence in the provisional Sapelovirus genus. Insects and skunk derived viral sequences exhibited amino acid identities ranging from 25% to 98% to the segmented genomes of viruses within the Reoviridae family. Two isolates were highly divergent: one is potentially a new species within the orthoreovirus genus, and the other is a new species within the orbivirus genus. We demonstrate that a simple, inexpensive, and rapid metagenomics approach is effective for identifying known and highly divergent new viruses in homogenized tissues of acutely infected mice.     

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。