A repetitive DNA sequence, rhs, responsible for duplications within the Escherichia coli K-12 chromosome.

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA-dependent. The rhsA locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhsA are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhsA or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed "transductional walking", by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.

Exchange of spacer regions between rRNA operons in Escherichia coli.

The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

Merodiploidy in Escherichia coli-Salmonella typhimurium crosses: the role of unequal recombination between ribosomal RNA genes.

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.

Effects of chromosomal inversion on cell fitness in Escherichia coli K-12.

In an effort to learn what factors might mitigate the establishment of Escherichia coli variants bearing major chromosomal rearrangements, we have examined the effects on cell growth of two inversions between rRNA operons. One of these inversions, IN(rrnD-rrnE), had been propagated in a commonly used subline of E. coli K-12 for approximately 30 yr before its discovery, a fact that illustrates the absence of obvious detrimental effects associated with the inversion. We found that culturing under conditions requiring repeated transition from stationary phase to rapid growth led to the replacement of IN(rrnD-rrnE) cells by cells that had undergone either of two types of additional chromosomal inversion: one type fully restored the wild-type order, while the other partially restored it. The partial reinversion was also between rrn operons, but it left a small transposition. The tendency for overgrowth by these revertants persisted through several rounds of periodic selection. In contrast, the other inversion, IN(rrnG-rrnE), was associated with severe, detrimental effects. The effects of IN(rrnG-rrnE) were also alleviated by full or partial reinversion. The probable relationship between the severity of the effects caused by the inversions and the degree of displacement of the replication origin is discussed. Spontaneous inversion events between rrn operons separated by 18% of the chromosome were estimated to occur at a frequency of roughly 10(-5). If extended to natural situations, the growth disadvantage together with the relatively high frequency of reinversion suggest that clones of cells with an inversion between these rrn operons would be readily overgrown by revertants.

Correlation of Rhs elements with Escherichia coli population structure.

The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.

Mosaic structure of plasmids from natural populations of Escherichia coli.

The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli. Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization. To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes. The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves. This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR. Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes. The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources. Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange. The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated.

Protein deprivation in primates. V. Fetal mortality and neonatal status of infant monkeys born of deprived mothers.

Seventy-four pregnancies were followed in 45 rhesus monkeys. While pregnant the mothers were fed a semisynthetic diet affording 1, 2, or 4 g protein/kg per day. The poorer diets tended to produce excess fetal mortality, but liveborn infants born to 1-g and 2-g mothers averaged as large as those in the 4-g group. No differences existed in total protein concentration and albumin-globulin ratios in the sera of newborn infants.

Protein deficiency in primates. IV. Pregnant rhesus monkey.

Young adult female rhesus monkeys, maintained in a seminatural environment, when pregnant 30 days were fed a balanced semisynthetic diet containing 15.3 percent casein (13.4 percent protein) or 0.5, or 0.25 that amount for the remainder of thir pregnancy. The diets, made isocaloric by the addition of carbohydrates to replace the missing casein, supplied 4, 2, or 1 g protein/kg per day if the animals ate 120 kcal/kg per day, an assumption that was reasonably accurate. We studied the responses of monkeys delivering normal young at term. All monkeys were clinically healthy during the experiment. All reduced their total plasma protein concentration, principally by decreasing their albumin concentration. Greatest loss occurred in the low-protein group. Albuminuria was a common finding in all groups. Food consumption though varying throughout pregnancy, remained fairly comparable from group to group. Weight gain of the highest protein group was greatest; that for the lowest protein group barely covered the weight of the products of conception. Blood pressure declined during pregnancy while blood glucose rose. There were no morphologic changes in hair bulbs and only suggestive changes in liver cells that pointed to increased vacuolation. Increased metabolic efficiency during pregnancy enables the mother to safely pass through a long pregnancy during most of which she is fed a low-protein diet. Nonpregnant aminals suffer more under the same circumstances.

Large genomic sequence repetitions in bacteria: lessons from rRNA operons and Rhs elements.

The rrn operons and Rhs elements provide starkly contrasting examples of the evolution and interaction of large sequence repetitions in bacteria. Genomic sequencing of different species as well as comparative sequencing of independent isolates is providing provocative insights into previously obscure issues.

Bilateral intraocular Nocardia asteroides infection.

A 38-year-old man with hypogammaglobulinemia and pulmonary Nocardia asteroides infection developed an intraocular Nocardia infection. The diagnosis was confirmed by examination of a specimen removed at pars plana vitrectomy. The chorioretinal infection in one eye resolved partially, with no organisms visible on histopathologic examination of the globe at autopsy. One month before the patient's death from disseminated nocardial infection, the previously uninvolved right eye developed a new metastatic nocardial chorioretinal lesion. This lesion rapidly progressed in size until the patient's death and showed on postmortem examination the presence of organisms characteristic of N asteroides.

Acrodermatitis enteropathica. Corneal involvement with histochemical and electron micrographic studies.

A case of acrodermatitis enteropathica occurred with corneal changes consisting of linear subepithelial corneal opacities. From the results of histochemical and electronmicrographic studies on a biopsy specimen of the corneal lesion, no definite conclusion may be drawn as to whether the corneal changes are a manifestation of acrodermatitis enterophathica or a result of iodochlorhydroxyquin therapy.

Differential effects of neuropeptides on short-term memory in primates.

In a within-subject design, six rhesus monkeys (3 males and 3 females) received a 100 micrograms/kg injection of one of seven neuropeptides or a diluent control solution and were then tested for activity level, learning (discrimination reversal), short-term memory (delayed response), and for responsiveness to noxious stimuli. One daily injection was made with a different peptide for 10 consecutive days, including pre- and post tests on th first and last days with the diluent control. DSIP and D-Phe4-Met enkephalin seemed to produce some interference with short-term memory, while alpha-MSH showed some facilitation of it, as indicated by interactions of the peptides with the delay periods of 0, 15, or 30 sec. Sex differences were found in the learning task and the responsiveness to a noxious stimulus, suggesting the possibility of interactions between the peptides and endogenous hormones.

Retinal astrocytoma.

A 41-year-old man had a 6 x 6 x 5-disk diameter amelanotic tumor in the posterior fundus. The clinical and fluorescein angiographic appearance suggested a benign retinal vascular tumor, although amelanotic choroidal melanoma and retinoblastoma were diagnostic possibilities. An incisional 48-hour radioactive phosphorus (32P) uptake test was performed and the result showed an increased uptake over the tumor mass of 100% as compared to the control quadrants. The globe was enucleated and the pathologic diagnosis was isolated astrocytic glioma of the retina with minimal, if any, malignant potential. The highly developed vascular system of the tumor probably contributed to the false-positive test result.

Effects of an enkephalin analog on complex learning in the rhesus monkey.

Facilitation of the learning of a discrimination reversal task for a reward of food was found in rhesus monkeys after subcutaneous administration of a potent pentafluorinated enkephalin analog. (D-Ala2)-F5-Phe4-enkephalin-NH2. General activity, short-term memory, startle, and analgesia, however, were not significantly affected. In a within-subject design, each of 6 monkeys (3 males and 3 females) received each of 5 doses of the enkephalin analog (0.1, 1, 10, 100, and 1,000 microgram/kg). One daily injection was made for 7 consecutive days, including pre- and posttests on the first and last days with the diluent control. The enkephalin doses, with the exception of the 0.1 microgram/kg level, produced significantly faster learning than the diluent. Some sex differences were suggested by the data, but these effects are difficult to interpret. The results suggest that relatively small amounts of this analog given systematically can exert a reliable effect on a complex behavior such as reversal learning at doses devoid of opiate effects, due perhaps to enhanced cognitive flexibility rather than improvement in short-term memory or association formation.

Probability-reward preferences of rhesus monkeys.

The relative preferences of rhesus monkeys for reward probability versus amount were investigated with procedures which contrasted general experience with specific instructions, and evaluated the relationship between probability-amount combinations and preference strength. Four stimulus objects, each signifying a different combination of reward frequency and amount (100% with one unit; 50% with two units; 33% with three units; or 25% with four units), were presented in pairs, one pair per daily session, with trial schedules providing the same amount of reward within each set of 12 trials. In Phase A, 4 monkeys (Group 1) were tested on the six choice-pairs with no preliminary training. In Phase B, Group 1 was joined by an additional 4 monkeys (Group 2), and each of the tasks was preceded by a demonstration of the relevant stimuli, one at a time, together with their associated probabilities and amounts. Group 1 animals developed preferences during Phase A for the more frequently rewarded objects, which persisted into Phase B, whereas Group 2 animals showed no preferences. This result indicates that preliminary instructions concerning the reward combinations associated with stimulus objects can prevent the development of a preference for greater probability over greater amount of reward, but cannot extinguish it once it has been formed or reestablished within the context of a particular task.

Protein deprivation in primates: VI. Food preferences of adult rhesus monkeys maintained on low-protein diets.

Three groups of adult female rhesus monkeys, maintained on low-protein diets (.5 gm, 1 gm, and 2 gm protein per kg body weight, were compared with a control group (4 gm protein per kg body weight) on a food-preference task. Food responsiveness was assessed by presenting 8 small pieces of a certain food, equally spaced about the perimeter of a turntable attached to the home cage, and recording number of pieces taken, number of pieces eaten, and elapsed time for taking all 8 pieces. 21 different foods were used in sequence, 3 each from the following 7 categories: cheese, meat, vegetable, nut, cereal, fruit, and candy. Scores on all 3 measures were highly correlated, and the order of preference was generally the same for all groups. The 2 lowest-protein groups accepted more foods at the lower end of the palatability spectrum than did either the 4-gm or the 2-gm group. There was a tendency for the foods least preferred by the protein-deprived monkeys to be themselves high in protein. Thus, although protein deprivation appears to increase the catholicity of food preference, there is no corresponding increase in the relationship between palatability and protein content.

Scotopic thresholds of rhesus monkeys at different grating densities under normal and low-protein diets.

Scotopic thresholds of rhesus monkeys were determined over a range of luminance levels, from 1138 to 17.8 x 10(-9) cd/cm2, and grating densities, from .132 to 1.58 lines per cm. The effects of a low-protein diet on these thresholds were also investigated, and standard stimulus objects within the discrimination learning situation were employed. Obtained thresholds decreased systematically from about 350 to 20 x 10(-9) cd/cm2, and there were no significant differences due to dietary protein level.