Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

SummaryHelicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strainspecific restriction-modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock-treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.

The pyrBI operon of Escherichia coli K-12 encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase,-EC 2.1.3.2). Expression of this operon apparently is negatively regulated by the intracellular levels of UTP. To elucidate the regulatory mechanism in which UTP functions, the nucleotide sequence of the promoter-regulatory region of the pyrBI operon was determined and DNA fragments containing this region were transcribed in vitroQ These experiments revealed a p-independent transcriptional terminator (attenuator) located only 23 base pairs before the promoter-proximal end of the structural genes. Transcription, initiated upstream at either of two potential pyrBI promoters was efficiently ("-98%) terminated at this site, indicating that the regulation of pyrBI expression involves atten-. uation control. Additional features identified suggest a model for regulation in which the relative-rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator.In Escherichia coli K-12 and closely related bacteria, de novo synthesis of UMP is catalyzed by six enzymes encoded by six unlinked pyrimidine genes and operons (1-3). The expression of these genes and operons appears to be noncoordinately regulated by pyrimidine nucleotides (4), but little is known about the regulatory mechanisms involved. The pyrBI operon encodes the catalytic (pyrB) and regulatory (pyrl) subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) (3). Previous in vivo studies have indicated that pyrBI expression is negatively regulated over a several hundredfold range by the levels of a uridine nucleotide (4). Recent experiments using an in vitro coupled transcription-translation system have identified UTP as the principal pyrimidine regulatory effector of this operon (5). This result and the observation that ATCase synthesis is preferentially stimulated by sublethal concentrations of inhibitors of transcriptional elongation in Salmonella typhimurium (unpublished data) suggest that the rate of UTP-dependent transcription is involved in the regulation of pyr'BI expression. In addition, ATCase synthesis was shown to be selectively inhibited in. a hisT strain of S. typhimunium (6), in which the rate of translational elongation is' slowed (ref. 7; D. Palmer and S. Artz, personal communication). This result suggests that the rates of both transcription and translation, perhaps of a leader sequence preceding the pyrBI structural genes, are involved in regulation. The regulatory mechanism could be similar to the attenuation control mechanisms of amino acid biosynthetic operons (8).In this study we determined the nucleotide sequence of the promoter-regulatory region of the pyrBI operon of E. coli K-12 and characterized in vitro transcription of DNA fragments containing this reg...

The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.