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S Austin

Publications and source records attributed to S Austin.

At least 109 records · Page 6Linked to original sources
PubMed1986

Partition functions of unit-copy plasmids can stabilize the maintenance of plasmid pBR322 at low copy number.

The maintenance of plasmid pBR322 is highly unstable in a polA12 strain of Escherichia coli at 29 degrees C due to severely reduced copy number. Under these conditions, introduction of the par (partition) locus of plasmid P1 or the par (sop) region of F into pBR322 stabilizes it. A region with similar activity was detected in the P7 plasmid. The activity of the P1 par locus was dependent on the P1 parA gene product and was sensitive to par-specified incompatibility.

DNA Replication↗
PubMed1985

The partition functions of P1, P7, and F miniplasmids.

The partition regions of P1, P7, and F miniplasmids are discrete DNA sequences of about 3 kb in length that will promote accurate partition of hybrid plasmids independent of the source of replication functions or the position or orientation of the elements. Each of the par regions seems to be very similarly organized, with open reading frames for essential proteins and a terminal site which appears to be analogous to the centromere of eukaryotic cells. When cloned, these terminal sites exert incompatibility against their respective parent plasmids presumably because they can compete with the parent plasmids as substrates for partition. We have determined the complete DNA sequence of the P1 par region. In addition to the open reading frame for the essential parA protein (42-44 kd), the region contains a second open reading frame which could encode a 38-kd protein. The 2 large open reading frames appear to form an operon that is negatively regulated from a site adjacent to the promoter and responds to the par gene products in trans. Both this site and the downstream "centromere" site, incB, contain blocks of extremely AT-rich sequences, which are postulated to be binding sites for par proteins. The incB and upstream AT-rich regions both contain 20-bp imperfect inverted repeats. Further downstream from the minimal incB sequence (172 bp) lies an additional region which is essential for partition. The further analysis of the P1 par region should be greatly facilitated by the finding that it can function in cis to stabilize pBR322 vectors under conditions where the copy number of pBR322 is reduced.

Amino Acid Sequence↗
PubMed1984

The effect of improved nutrition on plasma prolactin concentrations and postpartum infertility in lactating Gambian women.

A substantial increase in food consumption of lactating Gambian women has been shown to be associated with a reduction in their plasma prolactin concentration. Women receiving food supplements during pregnancy as well as in lactation exhibited an even greater lowering of the postpartum plasma levels of this hormone. Consequently prolactin values of supplemented women returned more quickly to levels which allowed the resumption of menstrual and ovulatory activity. Concurrent measurements of the plasma concentrations of estradiol and progesterone in addition to prolactin allowed the calculation of prolactin values at which half the lactating women could be expected to have resumed menstruation and ovulation. These values were 1007 and 759 microU/ml, respectively. Dietary improvement during lactation alone resulted in these critical prolactin concentrations being reached 21 wk earlier than in nonsupplemented counterparts, while those receiving the extra food in both pregnancy and lactation showed a 35-wk shortening of postpartum amenorrhea and infertility.

Amenorrhea↗
PubMed1983

Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition.

Hybrids formed by insertion of the plasmid maintenance regions of P1 or F into a lambda delta att vector form stable unit-copy plasmids in their Escherichia coli host. They must therefore both be substrates for an accurate cellular partition apparatus that ensures that all daughter cells inherit a plasmid copy. Analysis of deletion mutants of both types of hybrid showed that, although the P1 and F plasmid maintenance regions differ in sequence and specificity, they are similar in general organization. Each contains an approximately 3 X 10(3) base-pair region that is essential for replication (rep) and an adjacent but separable 3 X 10(3) base-pair region that is essential for the stability of plasmid maintenance (par). Each par region is thought to specify the recognition of the plasmid as a substrate for equipartition. The deletion mutants provide sources of isolated rep and par sequences from both P1 and F DNA. These elements were then used to construct composite plasmids with novel combinations and arrangements of rep and par sequences. Heterologous constructions containing P1 rep and F par or F rep and P1 par sequences were maintained faithfully. We conclude that par regions are both necessary and sufficient to promote equipartition of replicating plasmid DNA. This activity is exerted only in cis but otherwise seems to be independent of the position or orientation of the par sequences within the DNA. Both P1 and F par regions include DNA sequences (incB of P1, incD of F) that we propose are analogues of the centromeres of eukaryotic chromosomes. The remaining portions of the par regions are known to encode protein products that, we believe, act at the inc sites. Extra copies of these inc sites appear to exert incompatibility by competition for the cellular partition apparatus.

Bacteriophage lambda↗
PubMed1983

Partition of unit-copy miniplasmids to daughter cells. II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts.

The stable maintenance of the unit-copy lambda-P1:5R miniplasmid is dependent on adjacent but separable replication (rep) and partition (par) regions of DNA derived from its P1 plasmid parent. The par region consists of an approximately 2.5 X 10(3) base-pair (kb) segment of DNA of which the terminal kb contains the plasmid incompatibility determinant incB. Two of the 14 lambda-P1:5R partition-defective point mutants isolated are amber (nonsense) mutants, showing that a plasmid-encoded protein is essential for proper partition. All of the Par- point mutants are complemented by the wild-type par region in trans. The complementing activity was shown to be an Mr 44,000 protein encoded by the end of the par region distal to incB. Deletion analysis showed that the incB sequence is essential in cis to the plasmid in order that the plasmid be receptive to the par protein. Thus incB appears to be the target site for par protein activity. We propose that the protein binds to incB, forming a complex that is recognized as a substrate for the cellular partition apparatus. The ability of a cloned incB sequence to compete for the par protein or for the cellular partition apparatus accounts for its activity as an incompatibility determinant. The existence of a plasmid-encoded par protein suggests a specific model for equipartition.

Bacteriophage lambda↗
PubMed1983

Two mini-F-encoded proteins are essential for equipartition.

The par region of mini-F is both necessary and sufficient to promote equipartition of plasmid copies to daughter cells. It is approximately 2.5 kb long and contains the coding sequences for two proteins, F1 (41 kDa) and F2 (37 kDa). We isolated 13 mutants of a phage lambda-mini-F hybrid that form unstable plasmids. Two of these putative Par- mutants are fully suppressible nonsense (amber) mutants. One of the amber mutants, par-41, eliminates the synthesis of F1, generating a large nonsense fragment of the protein. The other mutant, par-36, eliminates the synthesis of F2. Thus both proteins appear to be essential for plasmid equipartition.

Bacterial Proteins↗
PubMed1983

Excess energy intake promotes the development of hypoalbuminaemia in rats fed on low-protein diets.

1. A group of rats were given ad lib, a diet with a protein-energy: total energy (P:E) value of 0 . 03. Other animals received the same protein intake (g/kg body-weight per d) as this group, but had their energy consumption reduced to either 90, 80, 70, 60 or 50% of the ad lib. value. 2. The restricted growth rate of rats fed on the P:E--0 . 03 diet ad lib. has been shown to be due entirely to their insufficient protein consumption. In contrast, energy intake was far in excess of that required for maintenance and the limited amount of growth. 3. Carcass analysis demonstrated that some of the excess energy intake was stored as fat, but a greater part had been dissipated, presumably by diet-induced thermogenesis. 4. The plasma concentration of triiodothyronine (T3) was elevated in all animals consuming excess energy and was significantly related to both the total surplus and the amount of energy dissipated. 5. In the group of animals restricted to 50% of the ad lib. intake, energy rather than protein appeared to be the factor limiting growth. Energy intake was below estimated requirements for maintenance and was associated with values for plasma T3 that were lower than those found in well-fed control rats. 6. Although all the animals had similar protein intakes, plasma albumin concentration differed between the groups and was found to be inversely proportional to the energy intake. Thus it was lowest in animals receiving food ad lib. and rose to near normal values in the most-severely-restricted rats. 7. It is suggested that hypoalbuminaemia, and perhaps other features of protein deficiency, seen in animals fed on low-P:E diets may occur as an undesirable consequence of the metabolic response required to deal with excess energy consumption.

Adipose Tissue↗
PubMed1983

Dietary manipulation of plasma albumin concentration.

The variation in plasma albumin concentrations of rats fed a wide range of protein- and energy-deficient diets has been investigated. These changes have been correlated with simultaneous effects of the diets on growth rate, carcass composition, protein and energy balance, and plasma concentration of insulin corticosterone and triiodothyronine (T3). Although rats eating diets of low protein content and libitum developed hypoalbuminemia, those fed equivalent amounts of protein at low energy intakes had plasma albumin concentrations nearer, or equal to control values. Data from the balance study showed that for hypoalbuminemia to occur, not only had dietary protein to be inadequate, it was also necessary for energy consumption to be in excess of requirements. It is suggested that in protein-deficient animals, energy consumed in excess of the amount required for maintenance and the protein-limited growth rate initiates a metabolic response which results in inappropriate use of the meagre protein intake. This "dysadapted" state, in the rat, appears to be indicated by elevated plasma T3 values. The implication that kwashiorkor-like symptoms can be generated by a relative as opposed to a total energy excess is discussed in relation to the appearance of this disease in humans.

Animals↗
PubMed1983

Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli.

The 140-megadalton plasmids of Shigella flexneri serotypes 1, 3, and 5, in addition to the 120-megadalton plasmid of Shigella sonnei, are associated with virulence. The present study showed that a 140-megadalton plasmid is also associated with virulence in Escherichia coli. When these plasmids were cleaved with EcoRI or BamHI restriction endonucleases, considerable homology was evident in plasmids from S. sonnei strains, whereas only a few common fragments were observed among the S. flexneri and enteroinvasive E. coli plasmids. Nitrocellulose filter hybridization demonstrated that, despite variations in restriction sites, all these plasmids shared a considerable complement of homologous sequences. Minicell-producing strains were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Transmission electron microscopy of infected HeLa cells showed that minicells from invasive strains retained the invasive phenotype. Sixteen polypeptides were labeled when S. flexneri 5 minicells were incubated with [35S]methionine. Fourteen of these plasmid-coded polypeptides were associated with the outer membrane in invasive strains of S. flexneri 5, and nine polypeptides of similar molecular weight were labeled in the outer membrane of invasive strains of S. flexneri 3, S. sonnei, and E. coli. Seven of the S. flexneri 5 polypeptides were not labeled in a noninvasive strain which had sustained a large deletion in the virulence-associated plasmid, and none were labeled in minicells which no longer harbored this plasmid.

Animals↗
PubMed1983

Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L.

We have isolated two new classes of P1 miniplasmids, called lambda-P1:5R and lambda-P1:5L, by the in vivo extension of a cloned P1 fragment, EcoRI-5, which by itself is not capable of plasmid replication. The lambda-P1:5R plasmids contain EcoRI-5 plus a variable portion of the adjacent P1 EcoRI fragment 8. They have a copy number like that of P1 (about 1 per host chromosome), are faithfully segregated at cell division, and are subject to incompatibility exerted by either a single copy of P1 or a single copy of EcoRI-5. In contrast, the lambda-P1:5L plasmids contain EcoRI-5 and a portion of adjacent P1 DNA that includes at least P1 EcoRI fragments 15, 18, and 23 and a part of fragment 17. These plasmids have a copy number of about 15 per cell chromosome. Despite this they are segregated to daughter cells somewhat less faithfully than are lambda-P1:5R plasmids. They are sensitive to incompatibility exerted by a single copy of P1, but not to incompatibility exerted by a single copy of EcoRI-5. lambda-P1:5L plasmids are, however, sensitive to incompatibility exerted by multiple copies of EcoRI-5. These results show that the relative copy numbers of exerting and responding elements are important for the incompatibility phenotype and strongly suggest that lambda-P1:5L plasmids lack a repressor of replication that can be supplied in trans from P1 but not from EcoRI fragment 5. We suggest that P1 normally uses the 5R replicon and that the 5L replicon may be a backup system that ensures plasmid maintenance should the primary replication event fail to initiate.

Bacteriophage lambda↗
PubMed1983

Differences in nitrogen metabolism between protein-deficient and energy-deficient rats with similarly restricted growth rates.

Paired groups of rats whose growth rates were restricted to similar degrees by feeding either protein- or energy-deficient diets had very different patterns of nitrogen metabolism. The protein-deficient animals showed evidence of dysadaptation in that liver protein was much more reduced than in the energy-deficient ones, whereas total muscle protein was higher. Plasma albumin concentrations decreased only in animals fed the low protein diet; with energy restriction a small but significant rise in albumin was seen. Although plasma amino acid concentration fell in both protein- and energy-restricted groups, a distorted pattern, with a markedly raised non-essential:essential amino acid ratio was found only in the protein-deficient rats. These differences in nitrogen metabolism could not be explained on the basis of variations in the plasma concentrations of insulin, corticosterone or growth hormone. The relationship of these changes to the development of kwashiorkor in man, and the use of plasma albumin and amino acid as indicators of inappropriate internal distribution of protein, resulting in dysadaptation, are discussed.

Amino Acids↗
PubMed1983

Metabolic consequences of fasting during Ramadan in pregnant and lactating women.

In studies in a rural West African village it was observed that all lactating women and 90 per cent of pregnant women fasted throughout the period of Ramadan. The metabolic consequences of this fasting were studied by measuring serum glucose, free fatty acid, triglyceride, beta-hydroxybutyrate, alanine, insulin, glucagon and T3 levels at 0700 h and 1900 h in 22 pregnant, 10 lactating and 10 non-pregnant, non-lactating women. Results were also compared with overnight-fasted values obtained outside Ramadan. Values for the lactating women were not significantly different from the non-pregnant, non-lactating controls despite the additional metabolic stress of lactation. Ramadan-fasted (1900 h) glucose values from women in late pregnancy (3.01 +/- 0.11 mmol/l) were significantly lower than all other groups (P less than 0.01) and were 15 per cent (P less than 0.01) lower than overnight-fasted values from similar subjects. Ramadan-fasted free fatty acid and beta-hydroxybutyrate levels were significantly higher (P less than 0.05) and alanine values were significantly lower (P less than 0.05) in late than in early pregnancy. It is concluded that the phenomenon of 'accelerated starvation' occurs when women in late pregnancy fast during Ramadan. The possible consequences of this failure to maintain glucose homoeostasis are discussed with reference to the poor outcome of the actual pregnancies studied.

3-Hydroxybutyric Acid↗
PubMed1982

Genetic and physical map of a P1 miniplasmid.

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.

Base Sequence↗
PubMed1981

A novel role for site-specific recombination in maintenance of bacterial replicons.

If daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell division. We present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage P1. Asymmetric partition occurs, leading to a relatively high rate of loss of the plasmid from the growing population. In contrast, the wild-type P1 plasmid is maintained very efficiently in host cells. We show that this efficient maintenance is due to the presence of the loxP-cre site-specific recombination system present on the intact P1 plasmid. This system promotes rapid recombination between two loxP sites on dimer molecules, resolving them into monomeric substrates for proper partition. We suggest that bacterial replicons that are maintained with great accuracy in recombination-proficient cells might also encode high-efficiency recombination systems.

Base Sequence↗