DNA arrays of the entire set of Escherichia coli genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose. Ratios of the transcript levels for all 4,290E. coli protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained. The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells. The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways. Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and rpoS expression was similarly elevated. The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced. The hallmark features of E. coli cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress. A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated. This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest.

In eukaryotic cells, cohesion between sister chromatids allows chromosomes to biorient on the metaphase plate and holds them together until they separate into daughter cells during mitosis. Cohesion is mediated by the cohesin protein complex. Although the association of this complex with particular regions of the genome is highly reproducible, it is unclear what distinguishes a chromosomal region for cohesin association. Since one of the primary locations of cohesin is intergenic regions between converging transcription units, we explored the relationship between transcription and cohesin localization. Chromatin immunoprecipitation followed by hybridization to a microarray (ChIP chip) indicated that transcript elongation into cohesin association sites results in the local disassociation of cohesin. Once transcription is halted, cohesin can reassociate with its original sites, independent of DNA replication and the cohesin loading factor Scc2, although cohesin association with chromosomes in G 2 /M is not functional for cohesion. A computer program was developed to systematically identify differences between two ChIP chip data sets. Our results are consistent with a model for cohesin association in which (i) a portion of cohesin can be dynamically loaded and unloaded to accommodate transcription and (ii) the cohesin complex has preferences for features of chromatin that are a reflection of the local transcriptional status. Taken together, our results suggest that cohesion may be degraded by transcription.Dividing cells must ensure that their chromosomes are copied exactly once and that new cells receive exactly a single copy of each chromosome. Failure to do so can result in aneuploidy, an abnormal number of chromosomes that typically leads to developmental abnormalities or cell death. During DNA replication, sister chromatids are cohered and cohesion is maintained until the metaphase-to-anaphase transition (28, 42). Sister chromatid cohesion facilitates the biorientation of chromosomes along the metaphase spindle and resists the tendency of microtubules to prematurely separate chromosomes once bipolar attachments are established (38). Cohesin also contributes to DNA repair (37, 44) and condensation (15).In eukaryotic cells, cohesion is mediated by several evolutionarily conserved proteins. The complex itself is composed of two SMC (structural maintenance of chromosomes) subunits, Smc1 and Smc3, and two non-SMC subunits, Mcd1/Scc1 and Scc3. Together, these subunits form a large ring-shaped complex that is essential for cohesion between sister chromatids (14, 16). The loading of cohesin onto chromosomes in G 1 is dependent on the Scc2/4 complex (5, 40). Cohesion is established during DNA replication (42). Cohesion is dissolved at the metaphase-to-anaphase transition by separase, which cleaves Mcd1, resulting in the movement of sister chromatids into separate daughter cells (41,43). Although the molecular structure of cohesin has been well studied, exactly how this ring-shaped complex interacts with DNA remains unc...

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent K m of 6 M for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have ؊10 and ؊35 sequence elements typical of 70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 M gluconate, modestly induced by very low levels of gluconate (4 M), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of l-idonic acid in whichd-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnRoperon, which encodes l-idonate 5-dehydrogenase, 5-keto-d-gluconate 5-reductase, an l-idonate transporter, and an l-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of l-idonate; IdnD catalyzes a reversible oxidation ofl-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to formd-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of d-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, withl-idonate or 5-ketogluconate serving as the true inducer of the pathway. The l-idonate 5-dehydrogenase and 5-keto-d-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of l-idonate andd-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize l-idonate as the sole carbon and energy source, and as predicted, the ability ofidnD, idnK, idnR, andedd mutants to grow on l-idonate is altered.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro . Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.