The most important portion of the mammary gland development occurs postnatally, with distinct periods of intensive morphogenesis taking place between birth and puberty and during pregnancy and lactation. To characterize the differentiation process of mammary myoepithelial cells, we have studied the expression patterns of several smooth muscle phenotypic markers, including contractile proteins, a-smooth muscle-actin (a-SM-actin), smooth muscle myosin heavy chains (SM-MHC), and calponin; components of cell-extracellular matrix adherens junctions, phosphoglucomutase-related protein (PGM), vinculin variants, integrin subunits; and laminin variant chains in the developing rat mammary gland using immunofluorescence microscopy. a-SM-actin-and SM-MHC-positive cells were found first in newborn animals, while calponin, PGM and a1 integrin subunit began to be expressed in prepubertal animals (1.5 weeks). Vinculin, p1 and a3 integrin subunits were largely confined to the basal cell layer at all developmental stages examined with greater staining starting at 1.5 weeks. Meta-vinculin was identified only in myoepithelial cells of the lactating gland. yl laminin chain was present in the mammary gland basement membrane throughout development, while the p2 chain was revealed in 3-week-old animals and accumulated later in postpubertal animals (7 weeks). Similarly, p2 laminin chain was absent from the forming alveoli basement membrane in pregnant rats and started to accumulate in the lactating gland. In addition to temporal changes, we have observed spatial differences in the distribution of the phenotypic markers. Both in pre-and in postpubertal animals, a-SM-actinand SM-MHC-positive cells of the growing ductal ends contained low amounts if any of calponin, PGM, and p2 laminin chain. We conclude that during postnatal development, mammary myoepithelial cells progressively acquire a differentiated phenotype as revealed by the expression of various smooth muscle markers. Maturation of the myoepithelial cells is accompanied by upregulation of the smooth muscle integrin expression followed by accumulation of P2-containing laminin variant. Thus, changes in adhesion system parallel with the myoepithelial cell differentiation.D 1995 Wiley-Liss, Inc.

Five recombinant plasmids, pBK2646, pBK611, pRC3, pRC4 and pRC5, carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained. The sequence analysis of these plasmids revealed certain structural changes in the rpoB gene which specify corresponding alterations in the beta-subunit of RNA polymerase. Some functional properties of the corresponding mutant strains and their RNA polymerases have been investigated.

The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion G----T, leading to amino acid substitution Val146----Phe. This mutational change marks the second domain of the beta subunit involved in rifampicin binding.

No abstract

We have analyzed the interaction of monoclonal antibodies against Escherichia coli RNA polymerase with products of its limited proteolysis. Two major proteolytic fragments of molecular masses 107 and 43 kDa originate as a result of a single cleavage in the vicinity of the 980th amino acid residue. Anti-/3 subunit monoclonal antibody PYN-2 inhibiting RNA polymerase activity at the stage of RNA elongation reacts with an epitope located between the amino-terminus and the 50th amino acid residue of the /3 subunit. DNA sequencing has shown that the RNA polymerase mutation rpoB22 converts the Gln(l111) codon of the fi subunit gene into the amber codon. An epitope for the monoclonal antibody PYN-6 was located between the major site of proteolytic cleavage and Gln(lll1) of the b subunit. RNA polymerase Monoclonal antibody LimitedproteolysisEpitope mapping Amber mutation