Three point mutations have been found in the hormone-binding domain (HBD) of the human androgen receptor (hAR): one in the N-terminal end (Ile663Asn in a family with partial androgen insensitivity syndrome (PAIS)); one in the middle, (Leu820Val in a family with PAIS); and one in the C-terminal end (Pro903Ser, in a family with complete AIS). The positions 663 and 903 were the most terminal mutation sites in the HBD found to date. The three mutant hARs have been previously characterized biochemically in genital skin fibroblasts. In the family with the Leu820Val substitution, the mother and the grandmother were found to be carriers for the same mutation. To prove their pathogenicity, each of the three mutations has been reproduced in an hAR expression vector that was transfected into COS-1 cells. In COS-1 cells, the complexes from Pro903Ser and Leu820Val had: increased thermolability; increased dissociation rates; decreased affinity; and abnormal transactivation. There was a hierarchy in the severity of the mutations expressed in kinetic and transactivation assays that correlated with the severity of the clinical phenotype. The pathogenicity of the Pro903Ser and the Leu820Val mutations was thereby confirmed. In COS-1 cells, the AR with Ile663Asn had normal thermolability, normal dissociation rates, and normal transactivation, but a decreased affinity. Although this sequence alteration has only been found in a PAIS patient, its pathogenicity is not considered to be proven. More sensitive assays are needed for this purpose.

The human androgen receptor (hAR) is a ligand-activated, nuclear transcription factor. Mutations affecting the formation and/or action of the hAR cause androgen insensitivity syndrome (AIS). The majority of mutations identified to date are within the DNA- and hormone-binding domains; very few have been identified in the transactivational modulatory domain, encoded by exon 1. This work presents an analysis of exon 1 and the 5$ sp prime$-flanking region of the hAR in a set of subjects whose AIS was believed to be caused by a mutation within these regions. Six of twelve strains had a nonsense or frameshift mutation in exon 1; a seventh strain had two missense and one silent substitution; no mutations were identified in the remaining subjects. The two missense mutations were recreated, individually and together, in an hAR complementary DNA (cDNA) expression vector and expressed in heterologous COS-1 cells. Their pathogenicity could not be proven with the system and assays used. In addition, mRNA and protein levels were analyzed and correlated with the identified mutations and the subjects' phenotype.

The human androgen receptor (hAR) is a ligand-activated transcription factor, and like other nuclear receptors, consists of a N-terminal modulatory domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (LBD). Several missense mutations in the LBD cause androgen insensitivity syndrome (AI), a condition in XY individuals with absent or subnormal male primary and secondary sexual characteristics. On the other hand, abnormal expansion of a polyglutamine tract in the N-terminal domain of the hAR causes spinal and bulbar muscular atrophy (SBMA) which also affects males and causes milder forms of AI, in addition to adult-onset motor neuron degeneration and gradual wasting and weakening of the muscles of the limbs, face, throat, and tongue. However, it was not clear how and to what extent these mutations contribute to the clinical phenotype of the affected individuals. In order to investigate this matter, I used PCR site-directed mutagenesis to create plasmids expressing hARs with two pairs of missense mutations in the LBD (Val865Leu and Val865Met, and Arg839His and Arg839Cys), discovered in AI individuals with varying severity of the phenotype, and two abnormal expansions of the polyglutamine repeat discovered in SBMA patients (40 and 50 glutamines). I also synthesized plasmids expressing no glutamines (0 glutamines), 12 glutamines, or 20 glutamines in the same N-terminal region of the hAR. These plasmids were transiently expressed in heterologous cells (COS-1 and PC-3), and the mutant hARs were assayed for ligand binding, stability, and transactivational capacity. / In contrast to the findings by others (McPhaul et al., 1992; Marcelli et al., 1994), in some instances involving identical mutations, I consistently observed a correlation between the biochemical phenotype of the mutant hARs and the clinical phenotype of AI individuals; that is, the more severe receptor phenotype was associated with the more severe AI. These results support the hypothesis that hAR phenotype is the dominant factor in the development of the secondary sexual characteristics in normal and affected individuals. / I also observed a tight negative correlation between polyglutamine tract length and transactivational capacity. This suggests that polyglutamine modulates the activity of the hAR, and that hAR activity might be suppressed in various androgen-sensitive tissues (including motor neurons) in SBMA individuals, thereby contributing to the age of onset and/or progression of the disease, even if it cannot be the primary pathogenic agent of the disease.

Because of its perceived ability to predict future health and its relevance for family members, genetic information challenges the traditional justifications for medical confidentiality. This thesis examines the question whether a health care professional should have the discretion or a duty to breach confidentiality in order to inform a patient's relatives of their increased genetic risk. There is currently no exception to the statutory, common law and ethical duties of confidentiality for the non-consensual disclosure of genetic information to relatives. Precedents developed in the context of threats of harm and communicable diseases are of limited value. The law should not recognise the existence of a duty to warn in the context of genetics. As a last resort, health care professionals should be authorized, but not required, to disclose genetic risk information when there is a serious risk of preventable harm and when the potential harm of non-disclosure outweighs that of disclosure.

The major objective of this thesis was to determine the molecular basis of a "ligand-selective" mutant androgen receptor (AR) phenotype. Methyltrienelone (MT), a synthetic androgen, dissociates normally from this receptor but mibolerone (MB), another synthetic androgen, dissociates from it two-fold faster than normal. This mutant receptor was identified within genital skin fibroblasts (GSF) from two unrelated individuals with different degrees of androgen insensitivity (AI). Sequence analysis of the AR gene from both subjects revealed a G to A transition at nt 2969 in exon 6 that alters codon 813 from serine to asparagine (S813N). Transiently expressed hAR.S813N did not reproduce the mutant phenotype in several heterologous cells: COS-1, BHK, CHO or HeLa cells. In contrast, when AR free (R$ sp-$) GSF were used as host cells, MB-R.S813N complexes dissociated almost two fold faster than the controls (n = 4) while MT-R.S813N complexes dissociated normally. These results establish the G to A transition at nt 2969 as the cause of the ligand-selective phenotype. Such host-cell restricted expression of the mutant dissociation rate points to cell-specific factors that can suppress abnormal dissociation of A-R complexes. Host cell-restricted expression of the abnormal dissociation rates has also been observed for two other transiently expressed mutant AR, hAR.V865L and hAR.R839H (n = 3). / Expansion of the glutamine (gln) tract within the N-terminus of the AR causes spinal bulbar muscular atrophy (SBMA), a disease of motor neurons, but the mechanism of this neuropathology is unknown. To determine the effect of gln-tract expansion upon AR function, SBMA-associated mutant AR was transiently expressed and characterized in COS-1 cells. The androgen-binding parameters of the mutant receptor were normal, but it had decreased transactivation competence (50-66% of normal; n = 3). This abnormal transregulatory function may account for the expression of traits associated with minimal androgen insensitivity (MAI) that are variably expressed in the SBMA patients.

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SRY (Sex determining Region of Y chromosome) is the master gene initiating testis determination in mammals. To shed light on the molecular mechanism of SRY action during testicular differentiation, we examined the effects of TAT-HMG fusion protein on gonadal sex differentiation in culture. HMG is the DNA binding motif of SRY and "TAT" is a protein transduction domain. Each pair of CD1 mouse gonadal primordia at 11.5 days post coitum (dpc) was cultured with or without TAT-HMG dissolved in dimethyl sulfoxide (DMSO) up to 3 days. Immunocytochemical labeling and Real-time RT-PCR of Sry, Sox9 and Mis indicated that DMSO blocked testicular differentiation, Sertoli cell differentiation and testis cords formation, downstream of SRY. TUNEL showed a massive mesenchymal cell death, which might be responsible for disruption of testis cord formation. Treatment with TAT-HMG rescued Sertoli cell differentiation, probably by up regulation of Sry, but not testis cord formation or cell death.

Highly repetitive, non-protein-coding satellite DNAs are ubiquitous among eukaryotes. In some cases, these sequences make up entire chromosomes, and as much as half of most eukaryotic genomes. Currently, very little is known about the possible roles of satellite DNAs in genome function. In this study I have begun to address the critical issue of satellite DNA function through two different approaches. First, I have used quantitative-RTPCR to address transcriptional levels of three different satellites known to express transcripts in the male germ tissue of the jewel wasp, Nasonia vitripennis. Two of these satellites are located uniquely on a supernumerary (‘extra’), non-essential chromosome known as PSR (for paternal sex ratio), while the third satellite is located on a normal chromosome. These experiments are suggesting that all three of these satellites are not expressed at consistent levels across individuals, arguing against a functional role. Instead, this finding supports the longstanding view of satellites as truly parasitic agents, and their expression may be artifactual. Second, I initiated experiments to determine if conditional mutagenesis through RNA interference is possible; development of this method in the wasp male germ line would be an invaluable tool for further assessing the function of satellite expression. Specifically, I tested the ability of RNAi to deplete the wasp ortholog of cannonball, which plays a testis-specific role in sperm formation in other insects. These experiments resulted in a trend of lower cannonball levels, although non-significant, in RNAi-treated males. These findings suggest that RNAi may be a potentially effective method for conditional mutagenesis in this tissue, but will require further optimization.

Genomes usually work together to promote the fitness of the organism, but sometimes parts of the genome cause intragenomic conflict, and act selfishly in order to promote their transmission. An example of this conflict is a selfish B chromosome known as paternal sex ratio (PSR) in the jewel wasp Nasonia vitripennis. Transmitted solely to new progeny with the sperms hereditary material, PSR completely destroys the paternal genome during the first mitotic division of the newly fertilized embryo. This effect enhances transmission of the PSR chromosome because of the unique haplodiploid reproductive mode of Nasonia and other members of the hymenopteran insect group. Through transcriptomic analyses, our group recently discovered that the PSR chromosome expresses eleven transcripts in the wasp testis. A plausible hypothesis is that one or more of these transcripts play some role in paternal genome elimination. In this study I have begun to test this hypothesis by screening through a set of previously produced truncated versions of the PSR chromosome. Specifically, I used PCR in order to screen these truncated chromosomes for the presence of each of these PSR-specific transcripts. I could then correlate the level of genome elimination induced by each truncated PSR chromosome with the presence or absence of the expressed transcripts. My work has established that (i) three of the eleven transcripts are likely not involved in genome elimination; (ii) no single transcript alone causes genome elimination; (iii) the remaining eight of eleven transcripts are viable candidates for causing genome elimination; and (iv) it is likely that a sub-group of these transcripts may operate together to induce this effect. I discuss several models in which PSR-expressed RNA molecules could operate to cause genome elimination.

Since changes in chromosome morphology contribute to the knowledge of evolution as well as to chromosome dynamics, this study looks specifically at one chromosome compared in twelve different species of Perissodactyls: Equus caballus (ECA), E. przewalskii (EPR), Equus africanus somaliensis (EAF), E. asinus (EAS), E. hemionus onager (EHO), E. h. kulan (EHK), E. h. kiang (EKI), E. zebra hartmannae (EZH), E. grevyi (EGR), E. burchelli (EBU), Tapirus indicus (TIN), and Rhinoceros unicornis (RUN). While chromosome morphology studies have been done in some of the extant equids, none have followed the evolution of this chromosome, homologous to Equus caballus chromosome 20 (ECA20), which contains the major histocompatibility complex (MHC). The gene order on the chromosome arm homologous to human chromosome six in most Equidae is reversed with respect to the centromere in comparison to humans. Multicolor fluorescence in situ hybridization was used to show that four probes from ECA20 hybridized to ECA20 (control), SWA5, EAS8, EHO16, EHK14, EKI16, EZH10, EGR11, EBU13, TIN4, and one of RUN12, 14, 15, or 22. The order for the four genes in the horses, zebras, and rhinoceros were as follows: cen-EDN1-MHC-ITPR3-MUT. Hybridization to the ass and tapir chromosomes displayed a possible neocentromere formation. It is apparent the chromosome has gone through several morphological changes while undergoing speciation in the Equidae, yet the overall gene order is conserved.