Although intercellular communication within peripheral nerves is critical to the structure and function of the nervous system, it is incompletely understood. Drosophila peripheral nerves comprise motor and sensory axons bundled by peripheral glia (Schwann cells) and wrapped by perineurial glia (perineurium). I have shown that growth of the perineurial glia is controlled by signaling pathways involving six genes: push, which encodes a large Zn2+ finger containing protein; amn which encodes a putative neuropeptide; Axs, which is suggested to encode a G-protein coupled receptor; ine, which encodes a putative neurotransmitter/osmolyte transporter; eag, which encodes a potassium channel; and NF1, which encodes neurofibromin and is the Drosophila ortholog of the human gene responsible for Neurofibromatosis type1. I provide evidence that neurofibromin, in accordance with its role as a Ras guanosine triphosphatase activating protein (Ras GAP), acts to down regulate Ras activity to control perineurial glial growth. My work suggests that loss of neurofibromin leads to an increase in Ras activity in the peripheral glia that, in conjunction with loss of either Ine or Push, introduces a cell-nonautonomous signal that promotes growth of the perineurial glia. I have also found that Push does not act through Ras to control perineurial glial growth. My working hypothesis is that Amn acts through two separate pathways, one involving Push and the other involving neurofibromin, to inhibit perineurial glial growth. In this model, a separate pathway involving the substrate neurotransmitter of Ine promotes perineurial glial growth. I speculate that Ine may act to remove its substrate neurotransmitter from the extracellular space, thereby inhibiting the neurotransmitter from acting through its receptor to promote perineurial glial growth. Alternatively, Ine may control perineurial glial growth via its role as an osmolyte transporter. Eag may act to inhibit perineurial glial growth by repressing release of factors from the neurons or peripheral glia through maintaining these cells in a hyperpolarized state.

The oligomeric assembly of the lactose repressor has been studied by a series of deletion and extension mutants at the C-terminal sequence. Deletion of the presumed mini leucine zipper sequence at the C-terminus (-5 aa, -11 aa, -18 aa, and -32 aa) has resulted in a family of dimeric repressors, while deletion of the last four amino acids (-4 aa) does not affect the oligomeric state or function of the repressor. Furthermore, extension of the C-terminus with leucine zipper-forming sequences (G359V+5 and GCN/C) has been deduced to strengthen a subunit interaction in tetramers. The role of the presumed mini leucine zipper in tetramer formation for the lac repressor is therefore confirmed. In addition, a long-axis dimer (GCN/C-Y282D) is obtained from increasing the leucine zipper length and disrupting a presumably distinctive interface composed of Tyr$\sp{282}$ and Lys$\sp{84}$. The generation of this altered protein confirms the postulate that there are two distinctive subunit interfaces for the assembly of tetrameric repressor. Detailed characterization of these mutant repressors has provided insight into the relationship between structure and function in the lac repressor. The subunit communication upon inducer-binding in a tetrameric repressor is between the two side-by-side monomers and does not appear to involve monomers in end-to-end orientation. The lower operator-affinity observed for the deletion dimers compared to the tetramer is derived from a difference in the association process.

We have studied spontaneous mutations in a chromosomally-located, single copy HSV-1 thymidine kinase (TK) gene in a human AK 143 TK$ sp-$ cell line. We have used three anti HSV-1 thymidine kinase nucleotide analogues, namely Trifluorothymidine (TFT), Acyclovir (ACV) and DHPG (9-(1,3-dihydroxy-2-propoxy-methyl)guanine), to select for $TK sp-$ mutants. The spontaneous mutation rate for the TK gene was very high in this system. The mutation frequency was 5 $ times$ 10$ sp{-3}$ per cell per generation when Acyclovir (ACV) was used as a selective agent and 5 $ times$ 10$ sp{-4}$ per cell per generation when Trifluorothymidine (TFT) was used. The spontaneous mutation frequency dropped to 1 $ times$ 10$ sp{-5}$ per cell per generation when a combination of TFT plus ACV was used and to 1 $ times$ 10$ sp{-6}$ per cell per generation when a combination of TFT plus DHPG (9-(1,3-dihydroxy-2-propoxy-methyl)guanine) was used as selective agents.

The mouse multidrug resistance mdr gene family is composed of three closely related members termed mdr1, mdr2, and mdr3. Gene specific hybridization probes have been used to determine that the three mdr genes are linked on a chromosomal segment of 625 kilobases, with the gene order and orientation: (5$ sp prime$) mdr3 (3$ sp prime$)-(5$ sp prime$) mdr1 (3$ sp prime$)-(3$ sp prime$) mdr2 (5$ sp prime$). In independently derived cell lines, we observed that the emergence of multidrug resistance is linked to the overexpression of either and mdr1 or the mdr3 gene. The mdr1 gene has been isolated and its genomic organization determined: the gene spans 68 kilobases, is split into 28 exons encoding discrete predicted domains of the protein, and appears to originate from the duplication of an intron-containing ancestor gene. The mdr1 promoter has been cloned, sequenced, and the transcription start site localized. The transcriptional activities of 5$ sp prime$ deletion fragments from the mdr1 promoter fused to a reporter gene have been tested by transient transfection in a panel of mouse cell lines. These studies have allowed the identification of proximal and distal regions in the mdr1 promoter, containing basal and cell-specific transcriptional activities.

Individuals born with low renal reserves are predisposed to progressive renal disease and primary hypertension as adults. Experimental evidence for vitamin A (retinol) control of nephrogenesis have led some to propose that maternal vitamin A status during pregnancy is responsible for most of the inborn variation in nephron number in the general population. The extent of collecting duct (CD) arborization during development is a primary determinant of nephron endowment. In the present study, the hypothesis of direct retinoic acid (RA) effects on CD branching morphogenesis was tested using murine inner medullary collecting duct (mIMCD-3) cells. Data presented show that mIMCD-3 cells express retinoic acid receptors (RARs) which mediate up to 30-fold induction of reporter activity from RARE-luciferase in transient transfection. In deep collagen culture, the RAR-selective agonist TTNPB and physiological levels of all-trans RA dramatically increase the number and length of branching tubules formed compared to control. Although retinoid regulation of EGF-R is well established in other cell lines, quantitative PCR and [125-I] EGF binding analysis of retinoid-treated cells provide no evidence of RA regulation of EGF-R transcripts or numbers at the cell surface. However, the cancellation of RA effects by the EGF-R inhibitor tyrphostin AG1478 suggests participation of active EGF-R in downstream signaling pathways; the possibility of receptor cross-talk by RA transactivation of EGF-R is raised. mIMCD-3 is a retinoid-responsive cell line whose branching program is strongly induced by RA. Identification of retinoid targets may have both fundamental and applied relevance, offering potential insight to in vivo processes, and as basic technology supporting tubular engineering and potential artificial renal replacement therapy.

The autosomal dominant spinocerebellar ataxias (SCAs) are a clinically heterogeneous group of neurodegenerative diseases. Since the first description by Pierre Marie in 1893, the classification has been controversial. This is mainly due to the variety of symptoms observed and the inter- and intrafamilial variability in age of onset, clinical features, neuropathological and biochemical findings. Up to the beginning of 1993, two loci had been identified: one on the short arm of chromosome 6 and more recently a second locus assigned to the long arm of chromosome 12 in a large Cuban family. These loci are termed SCA 1 and SCA 2, respectively. / We have studied four large families from different ethnic backgrounds segregating an autosomal dominant form of SCA. A total of 266 individuals, including 65 affecteds, were ascertained. We found clinical similarities among the four families. All kindreds showed progressive cerebellar ataxia, with a mean age onset in the fourth or fifth decades of life. / We performed detailed clinical, genetic and linkage analyses in these families in order to assess the clinical and genetic heterogeneity. (Abstract shortened by UMI.)

Somatically heritable change in regional chromatin structure leading to transcriptional inactivation is observed in a variety of organisms. Euchromatic and heterochromatic domains have been proposed to be formed and maintained by the action of nonhistone chromosomal proteins that either bind directly to DNA or are members of chromatin binding protein complexes. The nonhistone chromosomal proteins M31 and M32 are candidate inducer or maintenance genes of repressed chromatin states in the mouse. I have investigated M31 gene organization and M31 and M32 gene expression to further examine the possible role of these genes in the formation or maintenance of heterochromatic domains. Investigation of the organization of the M31 gene reveals that at least five M31 transcripts are produced by alternative splicing of 9 exons and/or premature termination with polyadenylation. Several transcripts are present before cytologically visible heterochromatin is detected, suggesting that the products of these transcripts have a role in the formation of heterochromatic domains. M32 produces only one transcript whose tissue pattern of expression is similar to that of M31.

The gene designated Pom-1 is a new mouse gene identified in the region of the Gin-1 common proviral integration site. The origin of the Gin-1 region has been described elsewhere (Villemur et al., 1987). The novel Pom-1 gene is testis-specific and encodes two major RNA species of 1.0 and 1.2 kbp. Our data have revealed that there is no clear evidence to suggest that Pom-1 is involved in the tumorigenic pathway that resulted in the Gross Passage A Murine leukemia virus-induced thymomas. The pattern of expression of Pom-1 was studied by a time course Northern blot, the STAPUT cell separation technique and in situ hybridization. Analysis of mRNA from enriched populations of spermatogenic cells from adult testes and localization by in situ hybridization revealed that Pom-1 transcripts are most abundant in the round and elongated spermatids, although there is a weak expression in pachytene spermatocytes. Immunocytochemistry data have shown protein expression to be localized in the Golgi apparatus of pachytene spermatocytes (stages IX-XII), round spermatids (steps 5-8) and elongated spermatids (steps 9-15). / The Pom-1 gene is not homologous to any sequences present in the Genebank. Sequence analysis predicts a 6 kilodalton protein which is basic, lysine and arginine rich ($ sim$12%). It is also relatively rich in potential phosphoacceptor amino acids ($ sim$20%), mainly threonine and serine, several of which are located in phosphorylation consensus sequences. These results suggest a role for the novel Pom-1 gene in spermatogenesis.

The nature of origins of DNA replication in mammalians has yet to be elucidated. The localization of the initiation sites and the identification of the corresponding sequences and/or structures is important to understand the process of initiation at the molecular level. The objective of the research in this thesis is to localize the initiation site of DNA synthesis at a specific locus on the chromosomes of mammalian cells, and to characterize the minimal sequence requirements for the function of this origin of DNA replication in vivo. A genomic clone was isolated using ors12, a mammalian autonomously replicating sequence (812 bp) that was previously isolated by extrusion of African Green monkey (CV-1 cells) nascent DNA from active replication bubbles. Ors12 associates with the nuclear matrix in a cell cycle dependent manner, and is present at the centromeric region of six CV-1 cell chromosomes and that of a marker chromosome. This genomic clone was used to generate PCR primers in a region encompassing ors12, in order to amplify nascent DNA strands isolated from asynchronously growing CV-1 and African Green monkey primary kidney cells. A competitive PCR-based mapping methodology was used, and revealed that DNA replication initiates preferentially in vivo in a region colocalizing with ors12, providing evidence of its function as a bona fide chromosomal origin of DNA replication. / In an attempt to define the sequence or structural elements that are important for mammalian origin function, a panel of deletion mutants of ors12 (812-bp) was generated. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. A 215-bp internal fragment was identified as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and MA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22-bp and 16-bp long, respectively.

Retinoic acid (RA), the major biologically active form of vitamin A, is required for normal development of the mouse embryo. In particular, RA is necessary for the correct anteroposterior specification of the embryonic axis. Exposure to RA at certain stages of development leads to premature truncation of the vertebral column, accompanied by spina bifida. In contrast, embryos that are homozygous null for RARgamma do not exhibit these defects, indicating that this receptor specifically mediates this teratogenic effect. Differential display PCR and suppression subtractive hybridization were employed using the RARgamma null embryos in an attempt to identify downstream targets that may be involved in the formation of these abnormalities. As a result of this process, a full-length cDNA molecule encoding a novel member of the aldo-keto reductase family (AKR1A4) was cloned. Although RA does not regulate the expression of this gene, its developmental expression pattern and substrate specificity suggests a potential role for this enzyme in the protection of certain rapidly growing embryonic structures from harmful metabolites. / A precise level of RA signaling is also required for proper specification of vertebral identity. Exposure to excess RA during the early stages of gastrulation results in posterior homeotic transformations of several vertebrae. These transformations are correlated with shifts in the anterior boundaries of Hox expression. In contrast, the loss of functional RARs leads to anterior vertebral transformations. Although retinoic acid response elements have been identified in Hox promoters, it is likely that RA regulates the expression of some Hox genes through intermediary factors. Cdx1 is a homeobox-containing transcription factor that influences vertebral patterning in a manner similar to RARs, and is directly regulated by RA in the mouse embryo. Analysis of an allelic series of RARgamma/Cdx1 null mutant mice demonstrated that Cdx1 and RARgamma act synergistically to pattern certain cervical vertebrae. In addition, Cdx1 is required for the full effect of RA treatment on the vertebral column. However, Cdx1 does not mediate all of the effects of RARgamma as the incidence of a thoracic to cervical vertebral transformation is significantly higher in RARgamma/Cdx1 double mutants as compared to either single null mouse.