Use of Starvation Promoters To Limit Growth and Selectively Enrich Expression of Trichloroethylene- and Phenol-Transforming Activity in Recombinant Escherichia coli

Volume 61, no. 9, p. 3323: the title of the article should read as shown above. [This corrects the article on p. 3323 in vol. 61.].     

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.     

Deactivation of macrophage oxidative burstin vitro by different strains ofHistoplasma capsulatum

It was previously reported thatHistoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi,Candida albicans andCryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.     

Creating new restriction sites by silent changes in coding sequences

We present methods for identifying a useful type of DNA site--one that can be mutated to create a new restriction site within a coding region without changing the amino acid sequence. These "latent sites" are abundant--silent mutations creating one of 44 different 6-bp or 8-bp recognition sites were found at relatively high density, roughly one latent site per 9 bp, in the eleven genes tested. Our analysis suggests that site-directed mutagenesis can be used to refashion coding sequences at will for flexible analysis.     

Cold responses of high altitude populations

The literature on cold stress in permanent high altitude residents and sojourners in the Himalayas and Andes is reviewed. High altitude natives, as exemplified by Peruvian Quechua Indians, are relatively well protected from the cold by the efficient use of wool clothing. However, exposure to wet-cold and dry-cold conditions is present, both diurnally and seasonally. Basal metabolism in the native is slightly elevated over United States norms, and natives are able to maintain high levels of blood flow to the extremities during whole-body and local extremity cooling tests. There is suggestive evidence for a developmental pattern of acclimatization to cold, but definitive evidence for genetic tolerance to cold in the highland native is lacking.     

Relative responses of an X-ray-resistant hybrid cell-line and its parent line to X-irradiation, ultraviolet light, actinomycin D and cordycepin.

Using colony formation as an assay, a rat-mouse hybrid cell-line (HD1) and one of its parent lines (H4) have been studied as to their abilities to survive exposure to ionizing radiation, ultraviolet light, and the drugs actinomycin D and cordycepin. HD1 cells are more resistant than H4 to ionizing radiation, actinomycin D and cordycepin. Both cell lines respond similarly to ultraviolet light. When both cell-lines were co-treated with actinomycin D or cordycepin, the toxic effect of ionizing radiation was enhanced, whereas that of ultraviolet light (U.V.L.) was unchanged. The data suggest that RNA synthesis is more important immediately after irradiation with X-rays than with U.V.L. and that cells resistant to the toxic effect of ionizing radiation are also resistant to the toxicity induced by inhibitors of RNA synthesis.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.