The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the fl and j' subunits of Escherichia coli DNAdependent RNA potynierase and of their structural genes, yielding a continuous nucleotide sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the fl subunit, comprising 1342 residues, and the N-terminal sequence of the jl' subunit (1 76 residues).Elucidation of the transcription mechanism requires detailed knowledge of the active-center organization of RNA polymerase at the various stages of the RNA synthesis. This, in turn, can be obtained only after determining the primary and spatial structure of the enzyme.Earlier we had established the amino acid sequence of the x subunit of Escherichiu coli DNA-dependent RNA polymerase by resorting solely to the ordinary methods of protein chemistry [5]. In the case of the fl and p' subunits with their much higher molecular weights (= 155000 and z 165000, respectively) [6], such an approach could no longer suffice, in view of the difficulties in isolating and purifying the resulting fragments and in reconstituting the amino acid sequence via overlapping peptides [7].The progress in DNA sequencing methods and the possibility of using the genetic code to obtain information on the primary protein structure from the nucleotide sequences is an attractive way to circumvent such difficulties; although here, too, there are many pitfalls, requiring considerable caution to avoid possible sources of error.In the first place the mRNA can undergo processing, leading to erroneous deduction of the protein structure. Secondly, the protein itself can be processed. Thirdly, it is often difficult to recognize in the overall DNA structure the beginning of a structural gene. The criterion for this purpose is the presence of an initiating codon together with the adjacent sequences complementary to the 3' end of 16-S RNA [8,9]. Frequently more than one such combination can be found for one and the same protein. insertion) in the DNA sequence determination could lead to a completely erroneous amino acid sequence of the protein. Thus, primary structure determination of DNA cannot serve as a substitute for the direct sequencing of the protein.In view of this, we decided to utilize the methods of both protein and nucleotide chemistries, performing the parallel sequencing of the structural genes rpoB (jl subunit) and rpoC (jl' subunit) and of the corresponding proteins. Knowledge of the nucleotide sequence of the pertinent DNA segments would permit aligning of the peptide fragments from the protein analysis into an uninterrupted polypeptide chain. Such...

No abstract

В статье приведены данные об изменении функции оставшейся почки после нефрэктомии у живого родственного донора. Показано, что в раннем послеоперационном периоде скорость клубочковой фильтрации и клиренс креатинина резко снижаются, а через месяц после операции в единственной почке развивается компенсаторная гиперфильтрация. В более поздний период происходит вторичное ухудшение функции почки, которое регистрируется и через 12 месяцев после нефрэктомии.

Приводятся данные о влиянии бикарбонатных диализирующих растворов с уксусной и молочной кислотами на кислотно-основное состояние, электролитный состав крови, параметры системной гемодинамики и показатели оксидативного стресса при проведении программного гемодиализа.

E. coli RNA polymerase holoenzyme consists of two large subunits--8 and p' (molecular weights 155,000 and 165,000), of two identical a subunits (molecular weight 36,500), and of the initiation factor u (molecular weight 86,000). The complete amino acid sequence of the RNA polymerase a subunit was determined. According to the primary structure the polypeptide chain of the a subunit has a molecular weight of 36,512 and consists of 329 amino acid residues. In order to determine the primary structure of large -8 and p' subunits of E. coli RNA polymerase, the protein fragmentation and sequencing procedure was combined with sequencing of the corresponding structural genes, which facilitated the ordering of the peptide fragments obtained during sequencing of the polypeptide chains. In all, 4714 base pairs of the rpoBC operon were sequenced including the entire structural gene of the -8 subunit and an initial segment (528 base pairs) of the p' structural gene. The -8 subunit appeared to consist of 1342 amino acid residues from which molecular mass was calculated to be 15061 8.6 daltons. The spatial organization of DNA-dependent RNA polymerase was studied with the aid of chemical modification and limited proteolysis. Photochemical affinity modifications were used to find out subunits of the enzyme responsible for binding of the DNA template and the RNA product.