A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long ␤ subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding ␤. Long deletions mimicking ␤ from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40 -60-amino acid-long deletions spanning ␤ residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of ␤ inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.DNA-dependent RNA polymerases from eubacteria share a common subunit composition (1). The core RNAP 1 enzyme (subunit composition ␣ 2 ␤␤Ј) is catalytically proficient but is unable to initiate transcription on promoters. Binding of a subunit converts the core enzyme into a holoenzyme, which can recognize a specific set of promoters (2). The ␤ and ␤Ј subunits together constitute more than 80% of the core RNAP mass and jointly form the catalytic center of the enzyme (3, 4). RNAPs from eukaryotes and archaea have subunits that are homologous to ␤ and ␤Ј of eubacterial enzymes (5-7). The evolutionary conservation within the ␤/␤Ј lineages is limited to relatively short segments of primary sequence; each subunit has 8 -10 highly conserved segments. The amino-to carboxyl-terminal order of the conserved segments is invariant.The spacing between the conserved segments can vary even when subunits from closely related species are compared, due to an accumulation of insertions and deletions. There are several reasons why studies of such evolutionarily variable regions can shed light on RNAP structure and function. First, these regions may form docking sites for species-specific regulators of transcription (8, 9). Second, variable regions are likely to be surface-exposed and can therefore be used for affinity tagging of RNAP and transcription complexes (10). Third, variable regions often tolerate splits, allowing preparation of functional RNAP with relatively short ␤ and/or ␤Ј subunit fragments, dramatically facilitating mapping of protein-protein and protein-nucleic acid contacts during transcription (11,12).The focus of this report is an evolutionary hypervariable region in the C-terminal portion of Escherichia coli ␤Ј (amino acids 1141-1131)...

The influence of low and high pub gene expression on the initial stages of the differentiation of mouse embryonic stem cells into derivatives of ecto-, meso-, and endoderm in vitro was investigated. As follows from the results of a RT -PCR analysis, the expression of the vimentin, somatostatin, GATA 4, and GATA 6 genes, being the markers of endodermal differentiation, does not vary in both the cells with high pub gene expression and the cells with low pub gene expression, as well as in the corresponding control lines. The cells with high pub gene expression are characterized by an increase in the expression of mesodermal differentiation gene-markers (trI card, trI skel, c-kit, and IL-7), whereas the cells with low pub gene expression are specified by a decrease in their expression. According to the analyses carried out, the reverse is characteristic of the expression of ectodermal differentiation gene-markers (nestin, ≤-III tubulin, gfap, and th). Expression of these genes decreases in cell lines with high pub gene expression, whereas their expression increases with the decrease in pub gene expression. Hence, it is suggested that the variations in the pub gene expression in the embryonic stem cells influence significantly the mesodermal and ectodermal differentiation of these cells.

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The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones.