Interaction in vivo between strong closely spaced constitutive promoters.

By genetic recombination procedures we have constructed strains of Escherichia coli K12 wherein up to three strong promoters ostensibly could function simultaneously and independently in the formation of messenger RNA molecules which specify tryptophan synthase. In all such multiple promoter cases, enzyme production falls short of the levels predicted from summing the measured contributions of each promoter taken singly. Possible molecular explanations for this phenomenon, which can produce apparent interferences in enzyme production as high as 42%, are considered.     

Presence of closely spaced protein thiols on the surface of mammalian cells

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.     

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation

Summary The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 by direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the Kpnl restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung ⁺ but not in extracts from ung ^– E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet ⁺ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung ⁺ E. coli strain in comparison with an ung ^– strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.     

Genetic structure among closely spaced leks in a peripheral population of lesser prairie-chickens

We evaluated the genetic structure of birds from four closely spaced leks in a peripheral population of lesser prairie-chickens (Tympanuchus pallidicinctus). Analyses of molecular variance revealed significant genetic structuring among birds from different leks for six microsatellite loci (FST = 0.036; P = 0.002), but we found no genetic differentiation at the mtDNA control region. Significant deviations from Hardy-Weinberg revealed an excess of homozygote genotypes within each of the leks studied (FIS = 0.190-0.307), indicative of increased inbreeding. Estimates of relatedness using microsatellite data suggest that the genetic structuring among lesser prairie-chicken leks occurs in part because of a lek mating system in which males at some leks are related. Structuring may also be caused by stochastic effects associated with a historical decline in population size leading to small, semi-isolated leks and high site fidelity by reproductive males. Results from this study suggest that microspatial genetic structuring may occur in lek-mating bird species with low levels of dispersal.     

Chemical composition of five deciduous tree species in four-year-old,closely spaced plantations

Summary Contents of N, P, K, Ca, Mg, and Mn in aboveground tree components of five deciduous species were determined in closely-spaced (0.9×0.6 m) 4-year-old plantations growing on a river terrace site in the Ohio Valley region of western Kentucky (USA). Species evaluated were: a hybrid poplar, American sycamore, European alder, river birch and green ash. The only significant difference in dry weight of tree components was greater bolebark biomass of the hybrid poplar. Total aboveground elemental content of N, K, Ca, and Mn varied significantly for some species. N content of green ash was significantly lower and K content of the hybrid poplar and Mn content of European alder were significantly greater compared with the other species. Ca contents of the hybrid poplar, American sycamore and European alder were significantly greater than those of other species. Based on the relationship between biomass production and nutrient content of the harvested biomass, it seems that on this and comparable sites, river birch is a preferred species in view of the lower potential nutrient removals in the harvested biomass.The investigation reported in this paper (79-8-129) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.