Escherichia coli has an ability, rare among the Enterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes, gadA, gadX, gadW, and gadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protected E. coli from organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF-and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes, yhiD and hdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>10 8 CFU per ml). Densitydependent acid resistance does not require any demonstrable secreted factor and may involve cell contactdependent activation. These findings further define the complex physiology of E. coli acid resistance.

SummaryEscherichia coli survives pH 2 environments through an acid resistance (AR) system regulated by the transcriptional activator GadE. Numerous proteins control gadE at an upstream, conserved, 798 bp intergenic region. We show this region produces three transcripts starting at -124 (T1), -324/-317 (T2) and -566 (T3) bp from the gadE start codon. Transcriptional lacZ fusions to gadE promoter regions revealed P1 and P3 were active while P2 alone was not. However, pairing P3 with P2 activated P2 and increased expression 20-fold above P3 alone. The fusions were transferred to Salmonella, which lacks this AR system, and plasmid-borne E. coli-specific regulators EvgA, YdeO, GadE and GadX were introduced. Data revealed that YdeO and GadX activate P3, P2 and P3P2, while GadE autoactivates P1 and represses P3 and P3P2. The developing model indicates that different signals activate YdeO, GadX, or an MnmE-dependent regulator, which stimulate gadE transcription from the P3 and P2 promoters. Once made, GadE activates P1 and represses P3 and P2. The P1 region also enables efficient downstream transcription and translation of the P3 or P2 transcripts. Evidence indicates the entire 750 bp sensory integration locus is necessary for a versatile response.

Escherichia coli can survive pH 2 acid stress by using several acid resistance systems. The most efficient of these employs glutamate decarboxylase (GadA/GadB) to consume protons, and an antiporter (GadC) to exchange the intracellular decarboxylation product for external glutamic acid. Expression of the essential transcriptional activator of this system, GadE, is controlled by several regulators in a hierarchical fashion. In this study, two additional activators have been identified. The AraC-family regulators GadX and GadW, previously found to activate gadA/BC in vitro, are now shown in vivo to directly activate gadE expression, which, in turn, activates the gadA/BC genes. In vivo results using E. coli and Salmonella enterica show that these regulators actually have little direct effect on gadA and gadBC promoters. The numerous gadE induction pathways converge on a 798 bp control region situated upstream of the gadE promoter region. Deletions of this control region exposed the region between "798 and "360 nt (relative to the translational start) to be required for maximum gadE-lacZ expression in Luria-Bertani (LB) medium and to be the primary focus of GadX and GadW control. The GadE protein itself, which binds to three GAD box sequences present between "233 and "42 nt, helped activate GadE expression in LB, but only when the "798 to "360 region was absent. These regulatory regions and proteins appear to integrate a variety of physiological signals that forecast a need for GadE-dependent gene expression and acid resistance.

A transgenic approach in mice allows the functional interrogation of an odorant receptor locus in vivo and reveals characteristics of its monogenic and monoallelic expression.

The metabolism of copper by cultured fibroblasts from brindled male (Mobr/Y) and normal male ( + / Y ) mice was studied using radioactive copper ("Cu). Brindled fibroblasts accumulated more 64Cu than normal fibroblasts. The accumulation of "Cu by both brindled and normal fibroblasts was enhanced if the cells were preincubated in a coppersupplemented medium or if fetal calf serum was omitted from the medium. Brindled fibroblasts also retained more 64Cu on continued incubation. The greater accumulation might, therefore, represent an effect of reduced copper efflux. The demonstrated expression of the inherited defect in copper metabolism of brindled mice in cultured fibroblasts emphasizes the value of this model for further study of copper metabolism and its genetic control. 153

Three brothers with neurofibromatosis (NF) died of sarcoma. The proband tumor had malignant schwannoma. The histologic nature of the sarcoma in the other two brothers was not known, but it seemed likely to be similar to that of the proband. However, the sarcomatous lesions in these two siblings developed in the patients' right lower limbs which showed clinical and radiological evidence of anterior bowing of the tibia. Their mother and two other siblings have neurofibromatosis, but have not developed malignant tumors. The unaffected father died at age 61 of carcinoma of the common bile duct. An unaffected sister developed adenocarcinoma of both colon and lung. Although it is known that patients with NF are at a high risk of developing malignant sarcoma and second primary malignancies, it is not clear whether this association is genetically determined. The family we report has some of the features of a "cancer family" and suggests that sarcomatous transformation in patients with NF may be genetically influenced. This report raises the possibility that the presence of congenital malformation and exposure to irradiation may predispose patients with neurofibromatosis to develop malignant schwannoma.

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Background and Objective: The Arabian oryx (Oryx leucoryx) was hunted to near extinction in from the 1950s to the 1970s. Thus, the oryx ranks among the rarest mammals in the world. Qatar currently has ~800 oryx and has donated numerous oryx to breeding programs around the world. As such, Qatar may be a significant source of genetic diversity to the worldwide oryx population. The primary purpose of this project was to provide a genome sequence and Single Nucleotide Polymorphism (SNP) data in hopes it can be used for improving breading strategies by maintaining as much genetic diversity as possible. Methods: A DNA sample was provided from an oryx male in the Wabra Wildlife Preservation in Qatar. The animal's whole genome was sequenced using next-generation sequencing approach. After assembling the contigs, we utilized a 67bp kmer and ~2.4B paired 100bp (~80X coverage) reads from the Illumina HiSeq. These reads were distributed across libraries ranging in size from 300-1200bp for paired-end and 2000-5000bp for mate-pair libraries. Genome physical coverage by mate-pair libraries was approximately 15X. De novo gene prediction was conducted on scaffolds >500bp. SNPs were also detected. Results: Predicted genome size of ~3Gb (similar to other Mammals), a scaffold N50 of ~300kb, an assembly spanning ~2.5Gb of the genome which is likely >90% of euchromatic sequence. The sequence is distributed across 36,964 scaffolds greater than 500bp. We detected ~1M SNPs between parental alleles, which is significantly fewer than in other "rare" animals such as the giant panda. Conclusion: The initial analysis of polymorphisms suggests a relatively high level of inbreeding, and further study will be needed to clarify whether this is only in certain herds or a worldwide issue.