Kisspeptins are products of the Kiss1 gene, which bind to GPR54, a G protein-coupled receptor. Kisspeptins and GPR54 have been implicated in the neuroendocrine regulation of GnRH secretion. To test the hypothesis that testosterone regulates Kiss1 gene expression, we compared the expression of KiSS-1 mRNA among groups of intact, castrated, and castrated/testosterone (T)-treated male mice. In the arcuate nucleus (Arc), castration resulted in a significant increase in KiSS-1 mRNA, which was completely reversed with T replacement, whereas in the anteroventral periventricular nucleus, the results were the opposite, i.e. castration decreased and T increased KiSS-1 mRNA expression. In the Arc, the effects of T on KiSS-1 mRNA were completely mimicked by estrogen but only partially mimicked by dihydrotestosterone, a nonaromatizable androgen, suggesting that both estrogen receptor (ER) and androgen receptor (AR) play a role in T-mediated regulation of KiSS-1. Studies of the effects of T on KiSS-1 expression in mice with either a deletion of the ERalpha or a hypomorphic allele to the AR revealed that the effects of T are mediated by both ERalpha and AR pathways, which was confirmed by the presence of either ERalpha or AR coexpression in most KiSS-1 neurons in the Arc. These observations suggest that KiSS-1 neurons in the Arc, whose transcriptional activity is inhibited by T, are targets for the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the anteroventral periventricular nucleus, whose activity is stimulated by T, may mediate other T-dependent processes.

CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst", essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (approximately 1:250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91(phox) deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with gamma-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients.

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.

The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS 1 and CEN3. The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.

We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.

OBJECTIVE

To investigate the prevalence and correlates of recovery from Diagnostic and Statistical Manual version IV (DSM-IV) alcohol dependence by examining the past-year status of individuals who met the criteria for prior-to-past-year (PPY) dependence.

METHODS

Cross-sectional, retrospective survey of a nationally representative sample of US adults 18 years of age and over (first wave of a planned longitudinal survey).

METHODS

This analysis is based on data from the 2001-02 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC), in which data were collected in personal interviews conducted with one randomly selected adult in each sample household. A subset of the NESARC sample (total n = 43 093), consisting of 4422 US adults 18 years of age and over classified with PPY DSM-IV alcohol dependence, were evaluated with respect to their past-year recovery status: past-year dependence, partial remission, full remission, asymptomatic risk drinking, abstinent recovery (AR) and non-abstinent recovery (NR). Correlates of past-year status were examined in bivariate analyses and using multivariate logistic regression models.

RESULTS

Of people classified with PPY alcohol dependence, 25.0% were still classified as dependent in the past year; 27.3% were classified as being in partial remission; 11.8% were asymptomatic risk drinkers who demonstrated a pattern of drinking that put them at risk of relapse; 17.7% were low-risk drinkers; and 18.2% were abstainers. Only 25.5% of people with PPY dependence ever received treatment. Being married was associated positively with the odds of both AR and NR, and ethanol intake was negatively associated with both. Severity of dependence increased the odds of AR but decreased the odds of NR. The odds of AR (but not NR) increased with age and female gender but were decreased by the presence of a personality disorder. Treatment history modified the effects of college attendance/graduation, age at onset and interval since onset on the odds of recovery.

CONCLUSIONS

There is a substantial level of recovery from alcohol dependence. Information on factors associated with recovery may be useful in targeting appropriate treatment modalities.

BACKGROUND

Animal models that closely mimic clinical disease can be exploited to hasten the pace of translational research. To this end, we have defined windows of opportunity in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of prostate cancer as a paradigm for designing pre-clinical trials.

METHODS

The incidence of cancer, metastasis, and distribution of pathology were examined as a function of time in TRAMP mice. The expression of various markers of differentiation were characterized.

RESULTS

The TRAMP model develops progressive, multifocal, and heterogeneous disease. Each lobe of the prostate progressed at a different rate. Cytokeratin 8, E-cadherin, and androgen receptor (AR) were expressed during cancer progression but levels were reduced or absent in late stage disease. A distinct epithelial to neuroendocrine (ENT) shift was observed to be a stochastic event related to prostate cancer progression in TRAMP.

CONCLUSIONS

This study will serve as the basis for the rational design of pre-clinical studies with genetically engineered mouse models.

Repression of the yeast silent mating type loci requires cis-acting sequences located over 1 kb from the regulated promoters. One of these sites, a "silencer," exhibits enhancer-like distance- and orientation-independence. The silencer demonstrates both autonomous replication sequence (ARS) activity and a centromere-like segregation function, suggesting roles for DNA replication and segregation in transcriptional repression. Here we identify three sequences (A, E, and B) involved both in repression and in either ARS or segregation activity. The sequences are functionally redundant: no one is essential for complete transcriptional control, but mutations in any two inactivate the silencer. Surprisingly, elements E and B can each activate transcription from heterologous promoters, and E shows striking homology to several yeast upstream activation sequences. Therefore, sequences individually involved in replication, segregation, and transcriptional activation can, at the silencer, efficiently repress transcription.

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

Globally, cardiovascular disease will continue causing most human deaths for the foreseeable future. The consistent gender gap in life span of approximately 5.6 yr in all advanced economies must derive from gender differences in age-specific cardiovascular death rates, which rise steeply in parallel for both genders but 5-10 yr earlier in men. The lack of inflection point at modal age of menopause, contrasting with unequivocally estrogen-dependent biological markers like breast cancer or bone density, makes estrogen protection of premenopausal women an unlikely explanation. Limited human data suggest that testosterone exposure does not shorten life span in either gender, and oral estrogen treatment increases risk of cardiovascular death in men as it does in women. Alternatively, androgen exposure in early life (perinatal androgen imprinting) may predispose males to earlier onset of atherosclerosis. Following the recent reevaluation of the estrogen-protection orthodoxy, empirical research has flourished into the role of androgens in the progression of cardiovascular disease, highlighting the need to better understand androgen receptor (AR) coregulators, nongenomic androgen effects, tissue-specific metabolic activation of androgens, and androgen sensitivity. Novel therapeutic targets may arise from understanding how androgens enhance early plaque formation and cause vasodilatation via nongenomic androgen effects on vascular smooth muscle, and how tissue-specific variations in androgen effects are modulated by AR coregulators as well as metabolic activation of testosterone to amplify (via 5alpha-reductase to form dihydrotestosterone acting on AR) or diversify (via aromatization to estradiol acting upon estrogen receptor alpha/beta) the biological effects of testosterone on the vasculature. Observational studies show that blood testosterone concentrations are consistently lower among men with cardiovascular disease, suggesting a possible preventive role for testosterone therapy, which requires critical evaluation by further prospective studies. Short-term interventional studies show that testosterone produces a modest but consistent improvement in cardiac ischemia over placebo, comparable to the effects of existing antianginal drugs. By contrast, testosterone therapy has no beneficial effects in peripheral arterial disease but has not been evaluated in cerebrovascular disease. Erectile dysfunction is most frequently caused by pelvic arterial insufficiency due to atherosclerosis, and its sentinel relationship to generalized atherosclerosis is insufficiently appreciated. The commonality of risk factor patterns and mechanisms (including endothelial dysfunction) suggests that the efficacy of antiatherogenic therapy is an important challenge with the potential to enhance men's motivation for prevention and treatment of cardiovascular diseases.

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Here we show that Akt and Mdm2 form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, we demonstrate that the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation.

Members of the transforming growth factor-beta (TGF-beta) superfamily bind to two different serine/threonine kinase receptors, i.e. type I and type II receptors. Upon ligand binding, type I receptors specifically activate intracellular Smad proteins. R-Smads are direct substrates of type I receptors; Smads 2 and 3 are specifically activated by activin/nodal and TGF-beta type I receptors, whereas Smads 1, 5 and 8 are activated by BMP type I receptors. Nearly 30 proteins have been identified as members of the TGF-beta superfamily in mammals, and can be classified based on whether they activate activin/TGF-beta-specific R-Smads (AR-Smads) or BMP-specific R-Smads (BR-Smads). R-Smads form complexes with Co-Smads and translocate into the nucleus, where they regulate the transcription of target genes. AR-Smads bind to various proteins, including transcription factors and transcriptional co-activators or co-repressors, whereas BR-Smads interact with other proteins less efficiently than AR-Smads. Id proteins are induced by BR-Smads, and play important roles in exhibiting some biological effects of BMPs. Understanding the mechanisms of TGF-beta superfamily signalling is thus important for the development of new ways to treat various clinical diseases in which TGF-beta superfamily signalling is involved.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

When prostate cancers progress following androgen depletion therapy, there are currently few treatment options with only one, docetaxel, that has been shown to prolong life. Recent work has shown that castration-resistant prostate cancers (CRPCs) continue to depend on androgen receptor (AR) signaling which is reactivated despite low serum androgen levels. Currently available AR-targeted therapy, including GnRH agonists and antiandrogens, cannot completely shut down AR signaling. Several mechanisms that enhance AR signaling in an androgen-depleted environment have been elucidated. These include AR mutations that allow activation by low androgen levels or by other endogenous steroids, AR overexpression, increased local intracrine synthesis of androgens, and upregulation of tyrosine kinase pathways. This has led to the development of a number of novel agents targeting the AR signaling pathway, including more effective antiandrogens, inhibitors of CYP17, an enzyme required for androgen synthesis, inhibitors of 5alpha-reductase, inhibitors of HSP90 which protects AR from degradation, inhibitors of histone deacetylases which is required for optimal AR-mediated transcription, as well as inhibitors of tyrosine kinase inhibitors. Many of these strategies are currently being tested in clinical trials in CRPC.

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.

OBJECTIVE

Patients with hormone receptor-negative breast cancer generally do not benefit from endocrine-targeted therapies. However, a subset with androgen receptor (AR) expression is predicted to respond to antiandrogen therapies. This phase II study explored bicalutamide in AR-positive, estrogen receptor (ER), and progesterone receptor (PgR)-negative metastatic breast cancer.

METHODS

Tumors from patients with ER/PgR-negative advanced breast cancer were tested centrally for AR [immunohistochemistry (IHC)>> 10% nuclear staining considered positive]. If either the primary or a metastatic site was positive, patients were eligible to receive the AR antagonist bicalutamide at a dose of 150 mg daily. Clinical benefit rate (CBR), the primary endpoint, was defined as the total number of patients who show a complete response (CR), partial response (PR), or stable disease (SD)>> 6 months; secondary endpoints included progression-free survival (PFS) and toxicity. Correlative studies included measurement of circulating endocrine markers and IHC surrogates for basal-like breast cancer.

RESULTS

Of 424 patients with ER/PgR-negative breast cancer, 12% tested AR-positive. The 6-month CBR was 19% [95% confidence interval (CI), 7%-39%] for bicalutamide. The median PFS was 12 weeks (95% CI, 11-22 weeks). Bicalutamide was well-tolerated with no grade 4/5 treatment-related adverse events observed.

CONCLUSIONS

AR was expressed in 12% of patients with ER/PgR-negative breast cancer screened for this trial. The CBR of 19% observed with bicalutamide shows proof of principle for the efficacy of minimally toxic androgen blockade in a select group of patients with ER/PgR-negative, AR-positive breast cancer.

The beta1- and beta2-adrenergic receptors (betaARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these betaARs, which are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined beta2AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional beta1ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, beta2ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of beta(2)ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.

Androgen receptor (AR) is reactivated in castration-resistant prostate cancer (CRPC) through mechanisms including marked increases in AR gene expression. We identify an enhancer in the AR second intron contributing to increased AR expression at low androgen levels in CRPC. Moreover, at increased androgen levels, the AR binds this site and represses AR gene expression through recruitment of lysine-specific demethylase 1 (LSD1) and H3K4me1,2 demethylation. AR similarly represses expression of multiple genes mediating androgen synthesis, DNA synthesis, and proliferation while stimulating genes mediating lipid and protein biosynthesis. Androgen levels in CRPC appear adequate to stimulate AR activity on enhancer elements, but not suppressor elements, resulting in increased expression of AR and AR repressed genes that contribute to cellular proliferation.

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of beta-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat alpha-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt(max)) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt(min), was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, beta-adrenergic receptor (beta-AR) density, and beta-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-beta is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-beta/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting beta-AR signaling capacity.

We have used pedigree analysis to investigate the mitotic segregation of circular and linear DNA plasmids in Saccharomyces cerevisae. Circular ARS plasmids, which bear putative chromosomal replication origins, have a high segregation frequency and a strong bias to segregate to the mother cell at mitosis. The segregation bias explains how the fraction of plasmid-bearing cells can be small despite the high average copy number of circular ARS plasmids. Linear ARS plasmids do not show strong segregation bias, nor does the 2 mu ori-containing plasmid YEp 13, when it is present in strains containing intact 2 mu circles. In the absence of endogenous 2 mu circles, YEp 13 behaves like an ARS plasmid, showing a strong maternal segregation bias. The presence of a centromere on circular ARS plasmids eliminates segregation bias. We discuss a model for plasmid segregation, which explains these findings and the possible biological significance of mother-daughter segregation bias.

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.