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Biomedical subjects

A F Stewart

Publications and source records attributed to A F Stewart.

At least 37 records · Page 2Linked to original sources
PubMed2000

RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners.

The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5'-3' exonuclease/annealing protein pairs, RecE/RecT and Redalpha/Redbeta, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein-protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.

Bacterial Proteins↗
PubMed2000

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

Actins↗
PubMed2000

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia.

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.

Animals↗
PubMed2000

BAC trimming: minimizing clone overlaps.

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.

Arabidopsis↗
PubMed2000

Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia.

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.

Animals↗
PubMed2000

DNA cloning by homologous recombination in Escherichia coli.

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.

Base Sequence↗
PubMed2000

Breast cancer incidence highest in the range of one species of house mouse, Mus domesticus.

Incidence of human breast cancer (HBC) varies geographically, but to date no environmental factor has explained this variation. Previously, we reported a 44% reduction in the incidence of breast cancer in women fully immunosuppressed following organ transplantation (Stewart et al (1995) Lancet 346: 796-798). In mice infected with the mouse mammary tumour virus (MMTV), immunosuppression also reduces the incidence of mammary tumours. DNA with 95% identity to MMTV is detected in 40% of human breast tumours (Wang et al (1995) Cancer Res 55: 5173-5179). These findings led us to ask whether the incidence of HBC could be correlated with the natural ranges of different species of wild mice. We found that the highest incidence of HBC worldwide occurs in lands where Mus domesticus is the resident native or introduced species of house mouse. Given the similar responses of humans and mice to immunosuppression, the near identity between human and mouse MTV DNA sequences, and the close association between HBC incidence and mouse ranges, we propose that humans acquire MMTV from mice. This zoonotic theory for a mouse-viral cause of HBC allows testable predictions and has potential importance in prevention.

Animals↗
PubMed2000

Point mutation of bacterial artificial chromosomes by ET recombination.

Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.

Chromosomes, Artificial, Bacterial↗
PubMed2000

Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34, parathyroid hormone-related protein 1-36, and SDZ-parathyroid hormone 893.

Daily administration of parathyroid hormone (PTH) and PTH-related protein (PTHrP) peptides has been shown to increase bone mass and strength in animals and, for PTH, to increase bone mass in humans. Long-term direct comparison of multiple members of the PTH/PTHrP family in vivo has not been reported. We therefore selected three PTH/PTHrP molecules for direct comparison in vivo in an adult rat model of postmenopausal osteoporosis: PTH(1-34), PTHrP(1-36), and the PTH analog, SDZ-PTH 893 ¿Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1-34 [hPTH(1-34)]¿. A 6-month study was performed in which adult (6-month-old) vehicle-treated ovariectomized (OVX) and sham OVX rats were compared with OVX rats receiving 40 micrograms/kg per day of either PTH(1-34), PTHrP(1-36), or PTH-SDZ-893. Bone mass, as assessed by ash weight and densitometry, bone histomorphometry, biomechanical properties at trabecular and cortical sites, and indices of bone formation markedly increased in all three PTH/PTHrP peptide-treated groups as compared with controls. In general, this improvement followed a rank order of SDZ-PTH-893 > PTH > PTHrP. The adverse effect profile also was greatest with SDZ-PTH-893; these rats developed moderate hypercalcemia, marked renal calcium accumulation, and displayed a 13% mortality. These studies show that PTH(1-34), PTHrP(1-36), and PTH-SDZ-893 significantly and progressively increase bone mass and bone strength in this rat model of postmenopausal osteoporosis. The adverse effect profile correlates in general terms with efficacy. All three peptides show promise as skeletal anabolic agents. Further studies in humans will be required to define optimal efficacy-to-adverse effect ratios and relative efficacy for each peptide in human osteoporosis.

Animals↗
PubMed1999

Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances.

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm.

Base Sequence↗
PubMed1999

Simplified generation of targeting constructs using ET recombination.

ET recombination is a way to engineer DNA in Escherichia coli using homologous recombination. Here we develop the potential of ET recombination in two ways relevant to complex engineering exercises such as building gene targeting constructs. First, a targeting construct was made in a single step. Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic clone. Subsequently a complex targeting construct was created by ligation with a multifunctional cassette.

Animals↗
PubMed1999

The chicken HPRT gene: a counter selectable marker for the DT40 cell line.

We describe the cloning, characterisation and chromosomal mapping of the chicken hprt gene together with the construction of two counter selectable hprt-/- DT40 derived cell lines. One of these cell lines contains a stably integrated gene encoding a conditionally active cre recombinase and thus allows efficient manipulation of targeted loci by site-specific recombination. These cell lines will enhance the utility of the hyper-recombinogenic DT40 cell line as a system for the genetic analysis of cell autonomous functions in vertebrates and as a tool for mammalian chromosome engineering.

Amino Acid Sequence↗
PubMed1999

Rapid modification of bacterial artificial chromosomes by ET-recombination.

We present a method to modify bacterial artificial chromosomes (BACs) resident in their host strain. The method is based on homologous recombination by ET-cloning. We have successfully modified BACs at two distinct loci by recombination with a PCR product containing homology arms of 50 nt. The procedure we describe here is rapid, was found to work with high efficiency and should be applicable to any BAC modification desired.

Bacterial Proteins↗
PubMed1999

Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system.

Gene therapy studies require techniques that allow alteration of human genomic DNA sequences. Bacterial artificial chromosome cloning systems (BACs/PACs) bridge the gap between vectors with small inserts and yeast artificial chromosomes (YACs). We report the use of a second generation BAC vector, pEBAC, containing eukaryotic selectable markers and combining some of the best features of the BAC, PAC and HAEC systems, into which a 185 kb sequence containing the human beta-globin gene cluster was retrofitted. To permit the introduction of mutations corresponding to those causing human pathology, we have adapted an inducible homologous recombination system for use in E. coli DH10B cells, the host strain for BACs and PACs. Using this system, we have introduced PCR fragments carrying a selectable marker and a reporter gene downstream of the IVS-110 splicing mutation into a specific site within the beta-globin gene sequence. The use of this inducible system minimises the risk of unwanted rearrangements by recombination between repetitive elements and allows the introduction of relevant modifications or reporters at any specific sequence within BACs/PACs in E. coli DH10B cells.

Cloning, Molecular↗
PubMed1999

Papillary thyroid carcinoma metastatic to the pituitary gland.

Many malignancies may present with metastases to the pituitary gland. The association of thyroid carcinoma with pituitary metastases is, however, very rare. This report describes two patients in whom metastases from a papillary thyroid carcinoma to the pituitary gland resulted in panhypopituitarism with blunted endogenous thyrotropin (TSH) production following withdrawal of levothyroxine. Both required the use of recombinant human TSH prior to radioiodine therapy. Symptoms of hypopituitarism may be difficult to distinguish clinically from those of hypothyroidism in the setting of levothyroxine withdrawal. Clinicians should be aware of the clinical and biochemical manifestations of this rare association.

Carcinoma, Papillary↗