Syntaxin 3B is essential for the exocytosis of synaptic vesicles in ribbon synapses of the retina.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Dominance of the proximal coordinate frame in determining the locations of hippocampal place cell activity during navigation.

The place-specific activity of hippocampal cells provides downstream structures with information regarding an animal's position within an environment and, perhaps, the location of goals within that environment. In rodents, recent research has suggested that distal cues primarily set the orientation of the spatial representation, whereas the boundaries of the behavioral apparatus determine the locations of place activity. The current study was designed to address possible biases in some previous research that may have minimized the likelihood of observing place activity bound to distal cues. Hippocampal single-unit activity was recorded from six freely moving rats as they were trained to perform a tone-initiated place-preference task on an open-field platform. To investigate whether place activity was bound to the room- or platform-based coordinate frame (or both), the platform was translated within the room at an "early" and at a "late" phase of task acquisition (Shift 1 and Shift 2). At both time points, CA1 and CA3 place cells demonstrated room-associated and/or platform-associated activity, or remapped in response to the platform shift. Shift 1 revealed place activity that reflected an interaction between a dominant platform-based (proximal) coordinate frame and a weaker room-based (distal) frame because many CA1 and CA3 place fields shifted to a location intermediate to the two reference frames. Shift 2 resulted in place activity that became more strongly bound to either the platform- or room-based coordinate frame, suggesting the emergence of two independent spatial frames of reference (with many more cells participating in platform-based than in room-based representations).

Chromosomal control of the dominant suppression of {\it ras\/}-mediated transformation

The role of tumor suppressor function in the multistep process of carcinogenesis was studied in the human teratocarcinoma cell line PA-1. Early passage PA-1 cells ($<$P100) are preneoplastic while late passage ($>$P100) PA-1 cells are spontaneously transformed. Previous work demonstrated a causal role for the N-ras oncogene in the neoplastic transformation of this cell line and the gene was cloned. A clonal cell line established at passage 40 has been shown to suppress the neoplastic transformation potential of the PA-1 N-ras oncogene in gene transfer experiments. This phenotype has been termed SRT+ for suppression of ras transformation. A clonal cell line established at passage 63 is neoplastically transformed by the N-ras in similar gene transfer experiments and is regarded as srt$-$. Somatic cell hybrids were formed between the SRT+ cell and two different N-ras transformed srt$-$ cells. The results indicate that five of the seven independent hybrid clones, and all 14 subclones, failed to form tumors in the nude mouse tumor assay. Chromosomal analysis of rare neoplastic segregants which arose from suppressed hybrid populations demonstrate that the general loss of chromosomes correlates with the reemergence of neoplastic transformation. Karyotype analyses demonstrate a statistically correlative loss of chromosomes 1, 4, 19, and to a lesser extent 11, 14, and 16. DNA hybridization analysis demonstrates a single copy of the intact N-ras oncogene in parental cells, suppressed hybrids, and neoplastically transformed hybrids. These results indicate that functional ras transformation suppression is a trans-dominant trait which may be controlled by sequences residing on particular chromosomes in the human genome. Furthermore, the suppression of ras transformation results from a unique step in the multistep process of carcinogenesis that is different from the induction of immortality. Thus, the neoplastic process of the PA-1 cell line involves at least three steps: (1) induction of immortality, (2) activation of the N-ras oncogene, and (3) loss of tumor suppressor function. ^

The story of RP10: A positional cloning approach to the identification of an autosomal dominant retinitis pigmentosa gene

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that is the leading cause of inherited blindness worldwide. Characteristic features of the disease include night blindness, progressive loss of visual fields, and deposition of pigment on the retina in a bone spicule-like pattern. RP is marked by extreme genetic heterogeneity with at least 19 autosomal dominant, autosomal recessive and X-linked loci identified. RP10, which maps to chromosome 7q, was the fifth autosomal dominant RP locus identified, and accounts for the early-onset disease in two independent families. Extensive linkage and haplotype analyses have been performed in these two families which have allowed the assignment of the disease locus to a 5-cM region on chromosome 7q31.3. In collaboration with Dr. Eric Green (National Center for Human Genome Research, National Institutes of Health), a well-characterized physical map of the region was constructed which includes YAC, BAC and cosmid coverage. The entire RP10 critical region resides within a 9-Mb well-characterized YAC contig. These physical maps not only provided the resources to undertake the CAIGES (cDNA amplification for identification of genomic expressed sequences) procedure for identification of retinal candidate genes within the critical region, but also identified a number of candidate genes, including transducin-$\gamma$ and blue cone pigment genes. All candidate genes examined were excluded. In addition, a number of ESTs were mapped within the critical region. EST20241, which was isolated from an eye library, corresponded to the 3$\sp\prime$ region of the ADP-ribosylation factor (ARF) 5 gene. ARF5, with its role in vesicle transport and possible participation in the regulation of the visual transduction pathway, became an extremely interesting candidate gene. Using a primer walking approach, the entire 3.2 kb genomic sequence of the ARF5 gene was generated and developed intronic primers to screen for coding region mutations in affected family members. No mutations were found in the ARF5 gene, however, a number of additional ESTs have been mapped to the critical region, and, as the large-scale sequencing projects get underway, megabases of raw sequence data from the RP10 region are becoming available. These resources will hasten the isolation and characterization of the RP10 gene. ^

CHROMOSOMAL MAPPING, LINKAGE RELATIONSHIPS, AND EVOLUTIONARY STUDIES OF THE HUMAN ANONYMOUS DNA CLONE D1S1 (IN SITU HYBRIDIZATION, PRIMATES, GENE MAPPING, AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA, GENE DUPLICATION)

D1S1, an anonymous human DNA clone originally called (lamda)Ch4-H3 or (lamda)H3, was the first single copy mapped to a human chromosome (1p36) by in situ hybridization. The chromosomal assignment has been confirmed in other laboratories by repeating the in situ hybridization but not by another method. In the present study, hybridization to a panel of hamster-human somatic cell hybrids revealed copies of D1S1 on both chromosomes 1 and 3. Subcloning D1S1 showed that the D1S1 clone itself is from chromosome 3, and the sequence detected by in situ hybridization is at least two copies of part of the chromosome 3 copy. This finding demonstrates the importance of verifying gene mapping with two methods and questions the accuracy of in situ hybridization mapping.^ Non-human mammals have only one copy of D1S1, and the non-human primate D1S1 map closely resembles the human chromosome 3 copy. Thus, the human chromosome 1 copies appear to be part of a very recent duplication that occurred after the divergence between humans and the other great apes.^ A moderately informative HindIII D1S1 RFLP was mapped to chromosome 3. This marker and 12 protein markers were applied to a linkage study of autosomal dominant retinitis pigmentosa (ADRP). None of the markers proved linkage, but adding the three families examined to previously published data raises the ADRP:Rh lod score to 1.92 at (THETA) = 0.30. ^

Traditional linkage analysis in admixed families

Currently there is no general method to study the impact of population admixture within families on the assumptions of random mating and consequently, Hardy-Weinberg equilibrium (HWE) and linkage equilibrium (LE) and on the inference obtained from traditional linkage analysis. ^ First, through simulation, the effect of admixture of two populations on the log of the odds (LOD) score was assessed, using Prostate Cancer as the typical disease model. Comparisons between simulated mixed and homogeneous families were performed. LOD scores under both models of admixture (within families and within a data set of homogeneous families) were closest to the homogeneous family scores of the population having the highest mixing proportion. Random sampling of families or ascertainment of families with disease affection status did not affect this observation, nor did the mode of inheritance (dominant/recessive) or sample size. ^ Second, after establishing the effect of admixture on the LOD score and inference for linkage, the presence of induced disequilibria by population admixture within families was studied and an adjustment procedure was developed. The adjustment did not force all disequilibria to disappear but because the families were adjusted for the population admixture, those replicates where the disequilibria exist are no longer affected by the disequilibria in terms of maximization for linkage. Furthermore, the adjustment was able to exclude uninformative families or families that had such a high departure from HWE and/or LE that their LOD scores were not reliable. ^ Together these observations imply that the presence of families of mixed population ancestry impacts linkage analysis in terms of the LOD score and the estimate of the recombination fraction. ^

Investigating the pathogenicity of mutations in two ubiquitously expressed housekeeping genes that cause autosomal dominant retinitis pigmentosa

The purpose of this dissertation research was to investigate potential mechanisms through which mutations in two ubiquitously expressed genes, inosine monophosphate dehydrogenase 1 (IMPDH1) and pre-mRNA processing factor 31 (PRPF31), cause autosomal dominant retinitis pigmentosa (adRP) but have no other apparent clinical consequences. Basic properties of the gene and gene product, such as expression and protein levels, were examined. The purpose of our research is to understand the genetic basis of inherited retinopathies such as retinitis pigmentosa (RP). RP is a heterogeneous retinal dystrophy that affects approximately one in 3,700 individuals, making it the most common heritable retinal degenerative disease worldwide. Currently, mutations in 35 genes are known to cause RP and additional loci have been mapped but the underlying gene is not yet known. Often the genes associated with RP are integral to the biological processes underlying vision, making their role in retinal disease easy to explain. However, the mechanisms by which other genes cause RP are not apparent, especially widely-expressed genes. For IMPDH1, this research characterized the enzymatic properties of retinal isoforms. Results show that the retinal isoforms have enzymatic functions similar to the previously known canonical IMPDH1 whether or not an adRP pigmentosa mutation is included in the protein. For PRPF31, this research tested the hypothesis that functional haploinsufficiency is the cause of disease and relates to nonpenetrance in some individuals. Studies in patients with known mutations show that haploinsufficiency is the likely cause of disease, however, we did not confirm that non-penetrant individuals are protected from disease via increased expression of the wild type allele. Information gleaned from these functional studies, and the testing methods developed in tandem, will contribute to future research on disease mechanism related to adRP. ^

Creation of a retinoblastoma mutant with dominant -negative activity

The Retinoblastoma tumor suppressor gene (RB) plays a role in a variety of human cancers. Experimental analyses have indicated that the protein product of the RB gene (pRb) plays a role in cell cycle regulation, and that this protein is required in cellular differentiation, senescence, and cell survival. pRb function is dependent on its ability to bind to cellular factors. There are multiple protein binding domains within pRb. Mutations within these domains which eliminate the ability of pRb to bind its targets result in loss of function. Loss of pRb function leads to tumorigenesis, although uncontrolled cellular proliferation is not a universal response to pRb inactivation. The ultimate response to the loss of pRb is influenced by both the genetic and epigenetic environments. Targeted disruption of RB in mice results in embryonic lethality, demonstrating the requirement for functional pRb in development. Close examination of various tissues from the embryos which lack wildtype RB shows problems in differentiation as well as showing induction of apoptosis. Although disruption of RB has provided useful information, complete inactivation of a gene precludes the possibility of discovering the functions that separate domains may have within the system. Creation of a dominant negative mutant by domain deletion whose phenotype is expressed in the presence of the wildtype may provide information about the intermediate functions of the protein. In addition, tissue specific targeting of a dominant negative mutant of pRb allows for comprehensive analysis of pRb function in organogenesis. In this thesis, a series of RB deletion mutants were created and tested for dominant negative activity as well as cellular localization. A tissue culture assay for dominant negative activity was developed which screens for the phenotype of apoptosis due to loss of pRb function. Two mutants from this series scored positive for dominant negative activity in this assay. The effect of these mutants within the assay environment can be explained by a model in which pRb acts as a facilitator of cell fate pathway decisions. ^

Signal transduction of {\it HER-2\/}/{\it neu\/} receptor tyrosine kinase

Overexpression and/or amplification of HER2/neu is frequently detected in many human cancers. Activation of p185 tyrosine kinase can be achieved by point mutation, overexpression, deletion, and heterodimerization with other class I receptors. In this study I investigated the signal transduction pathways mediating the oncogenic signal of the point mutation-activated rat p185. I demonstrated that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex correlated to the transformation of NIH3T3 cells caused by the point mutation-activated rat HER2/neu. Furthermore, I observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated p185. The truncated p185 product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. These results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role in cell transformation induced by the point mutation-activated p185, and that stable binding to mutant p185 may not be necessary for Shc to mediate this signaling pathway.^ Recent studies have suggested that formation of the complex containing Sos, Grb2 and Shc is important in coupling receptor tyrosine kinases to the Ras signaling pathway. To clarify the role of this trimer in the oncogenic signaling of the activated p185, I set out to interfere with the protein-protein interactions in Shc/Grb2/Sos complex by introducing Grb2 mutants with deletions in either amino- ($\Delta$N-Grb2) or carboxyl- ($\Delta$C-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells that express the point mutation-activated HER-2/neu. I found that the transformed phenotypes of the B104-1-1 cells were largely reversed by expression of the $\Delta$N-Grb2. The effect of the $\Delta$C-Grb2 on phenotypic reversion was much weaker. Biochemical analysis showed that the $\Delta$N-Grb2 was able to associate Shc but not the activated p185 nor Sos, while the $\Delta$C-Grb2 bound to Shc, the activated p185, and Sos. The p185-mediated Ras activation was severely inhibited by the $\Delta$N-Grb2 but not the $\Delta$C-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the $\Delta$N-Grb2 is able to impair the oncogenic signaling of the mutation-activated p185, indicating that (i) the $\Delta$N-Grb2 functions as a strong dominant-negative mutant, (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the mutation-activated p185. Unlike the $\Delta$N-Grb2, the $\Delta$C-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild-type Grb2. ^