Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

NHERF1 – New Modifier of Colorectal Cancer Progression

Colorectal cancer (CRC) develops from multiple progressive modifications of normal intestinal epithelium into adenocarcinoma. Loss of cell polarity has been implicated as an early event in this process, but the molecular players involved are not well known. NHERF1 (Na+/H+ Exchanger Regulatory Factor 1) is an adaptor protein with apical membrane localization in polarized epithelia. In this study, we tested our hypothesis that NHERF1 plays a role in CRC. We examined surgical CRC resection specimens for changes in NHERF1 expression, and modeled these changes in two- and three-dimensional (2D and 3D) Caco-2 CRC cell systems. NHERF1 had significant alterations from normal to adenoma and carcinoma transitions (2=38.5, d.f.=4, P<0.001), displaying apical membrane localization in normal tissue but loss of expression in adenoma and ectopic overexpression in carcinoma. In Caco-2 cell models, NHERF1 depletion induced epithelial-mesenchymal-transition in 2D cell monolayers and disruption of apical-basal polarity in 3D cyst system. The mesenchymal phenotype of NHERF1-depleted cells was fully restored by re-expression of NHERF1 at the apical membrane. Cytoplasmic and nuclear NHERF1 re-expression not only failed to restore the epithelial phenotype but led to more aggressive phenotypes. Our findings suggest that membrane NHERF1 is an important regulator of epithelial morphogenesis, and that changes in NHERF1 expression correlate with CRC progression. NHERF1 loss and ectopic expression that induce massive disruption of epithelial cell polarity may, thereby, mark important steps in CRC development.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A

STUDY OF REST AS A NEGATIVE REGULATOR OF P16INK4A Monica Gireud, B.S. Thesis Advisor: Vidya Gopalakrishnan, Ph.D. The RE1 Silencing Transcription Factor (REST) is a negative regulator of neuronal differentiation. It is expressed ubiquitously in early embryos, but downregulated in neural progenitors concomitant with onset of neuronal differentiation in these cells. REST has been widely studied as a negative regulator of neuronal differentiation genes. Our recent work identified a novel role for REST in control of cell proliferation. However, the underlying molecular mechanism(s) are not known and is a focus of the current thesis project. Here, we provide evidence that REST signaling controls the expression of the cyclin-dependent kinase inhibitor, p16Ink4a, a negative regulator of the cell cycle and passage through G1. We determined that REST expression in the proliferating granule progenitors of the cerebellum and its lack of expression in the differentiated neurons is reciprocally correlated with that of p16Ink4a. Decline in REST levels in differentiating primary and neural stem cells immortalized with v-myc (NSC-M) granule progenitors in vitro was also associated with upregulation of p16Ink4a expression. Conversely, constitutive human REST transgene expression in NSC-M cells (NSC-MRs) blocked p16Ink4 upregulation, even under neuronal differentiation conditions. However, the lack of a consensus REST DNA binding RE1 element in the regulatory regions of p16Ink4a locus suggested an indirect regulation of p16Ink4a by REST. Based on work from other groups that showed repression of p16Ink4a transcription by the polycomb protein Bmi-1, and its negative regulation by microRNA-203 (miR-203) and our identification of a RE1 element in the downstream regulatory region of miR-203, we asked if the p16Ink4a expression was controlled by REST through a series of negative regulatory events involving miR-203 and Bmi-1. We observed that Bmi1 -expression mirrored that of REST and inversely correlated with that of miR-203 in the postnatal cerebellum and in vitro differentiated granule and NSC-M progenitors. In contrast, forced REST transgene expression in NSC-MR cells abrogated the decrease in Bmi-1 levels and elevation in miR-203 expression. Significant REST binding to the miR-203 RE1 element was also observed in NSC-M cells, indicating that REST had the potential to directly regulate miR-203 expression. In conclusion, our studies suggest a role for REST in control of cell cycle transit in neural progenitors through negative regulation of p16Ink4a. Further validation of these results in REST knockout mice is needed, and is ongoing.

Isolation and analysis of regulatory and structural mutations in the {\it recA\/} gene of {\it Escherichia coli}

The recA gene is essential for homologous recombination and for inducible DNA repair in Escherichia coli. The level of recA expression is important for these functions. The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine of these mutations were single base changes in the recA promoter; each reduced both induced and basal (repressed) levels of expression, indicating that only one promoter is used under both circumstances. Deletion analysis of the promoter region and S1 mapping of transcripts confirmed that there is only one promoter responsible for both basal and induced expression. Some of the mutants, however, displayed a ratio of induced to repressed expression that was much lower than wild-type. For one of these mutants (recA1270) LexA binding studies showed that this was not due to a change in the affinity of LexA repressor for the operator site. The extent of binding of RNA polymerase to this mutant promoter, however, was much reduced, and the complexes formed were qualitatively different. Further binding experiments provided some evidence that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation. Behavior of the mutants with altered induction ratios could be explained if LexA binding to the operator actually increases RNA polymerase binding to the promoter in a closed complex compensating for defects in polymerase binding caused by the mutations.^ In a study of mutations in the recA structural gene, site-directed mutagenesis was used to replace cysteine codons at positions 90, 116, and 129 with a number of different codons. In vivo analysis of the replacements showed that none of the cysteines is absolutely essential and that they do not have a direct role as catalysts in ATP hydrolysis. Some amino acid substitutions abolished all RecA functions, while a few resulted in partial or altered function. Amino acids at positions 90 and 129 tended to affect all functions equally, while the amino acid at position 116 appeared to have a particular effect on the protease activity of the protein. ^

Analysis of a human placental lactogen gene promoter

Human placental lactogen (hPL) and human growth hormone (hGH) comprise a multigene family that share $>$90% nucleic acid sequence homology including 500 bp of 5$\sp\prime$ flanking sequence. Despite these similarities, hGH is produced in the anterior pituitary while hPL is expressed in the placenta. For most genes studied to date, regulation of expression occurs by alterations at the level of transcriptional initiation. Nuclear proteins bind specific DNA sequences in the promoter to regulate gene expression. In this study, the hPL$\sb3$ promoter was analyzed for DNA sequences that contribute to its expression. The interaction between the hPL$\sb3$ promoter and nuclear proteins was examined using nuclear extracts from placental and non-placental cells.^ To identify regulatory elements in the promoter of the hPL$\sb3$ gene, 5$\sp\prime$ deletion mutants were constructed by cleaving 1200 bp of upstream sequence with various restriction enzymes. These DNA fragments were ligated 5$\sp\prime$ to a promoterless bacterial gene chloramphenicol acetyltransferase (CAT) and transfected into JEG-3 cells, a human placental choriocarcinoma cell line. The level of CAT activity reflects the ability of the promoter mutants to activate transcription. Deletion of the sequence between $-$142 bp and $-$129 bp, relative to the start of transcription, resulted in an 8-fold decrease in CAT activity. Nuclear proteins from JEG-3, HeLa, and HepG2 (human liver cells), formed specific binding complexes with this region of the hPL$\sb3$ promoter, as shown by gel mobility shift assay. The $-$142 bp to $-$129 bp region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. Sp1-like proteins were identified by DNA binding assay, in the nuclear extracts of the three cell lines. A series of G nucleotides in the hPL$\sb3$ promoter regulatory region were identified by methylation interference assay to interact with the DNA-binding proteins and the pattern obtained is similar to that for other Sp1 binding sites that have been studied. This suggests that hPL$\sb3$ may be transcriptionally regulated by Sp1 or a Sp1-like transacting factor. ^

The development and characterization of two types of chronic responses in irradiated mouse colon

The hypothesis to be tested is that there are two distinct types of chronic responses in irradiated normal tissues, each resulting from damage to different cell populations in the tissue. The first is a sequala of chronic epithelial depletion in which the tissue's integrity cannot be maintained, i.e. a "consequential" chronic response. The other response is due to cell loss in the connective tissue and/or vascular stroma, i.e. a "primary" chronic response. The purpose of this study was to test the hypothesis in the murine colon by first, establishing a model of each chronic response and then, by determining whether the responses differed in timing of expression, histology, and expression of specific collagen types. The model of late damage used was colonic obstructions/strictures induced by a single dose of 27 Gy ("consequential" response) and two equal doses of 14.75 Gy (t = 10 days) ("primary" response). "Consequential" lesions appeared as early as 5 weeks after 27 Gy and were characterized by a deep mucosal ulceration and a thickened fibrotic serosa containing excessive accumulations of collagen types I and III. Both types were commingled in the scar at the base of the ulcer. Fibroblasts were synthesizing pro-collagen types I and III mRNA 10 weeks prior to measurable increases in collagen. A significant decrease in the ratio of collagen types I:III was associated with the "consequential" response at 4-5 months post-irradiation. The "primary" response, on the other hand, did not appear until 40 weeks after the split dose even though the total dose delivered was approximately the same as that for the "consequential" response. The "primary" response was characterized with an intact mucosa and a thickened fibrotic submucosa which contained excessive amounts of only collagen type I. An increased number of fibroblasts were synthesizing pro-collagen type I mRNA nearly 25 weeks before collagen type I levels were increased. The "primary" response lesion had a significantly elevated collagen type I:III ratio at 10-13 months post-irradiation. These data show a clear difference between the two chronic response and suggest that not all chronic responses share a common pathogenesis, but depend on the cell population in the tissue that is damaged. ^

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

{\it Hox\/} genes and specification of regional identity in the axial skeleton: Analysis of the individual and combined mutant phenotypes of the paralogous group 4 murine {\it Hox\/} genes; {\it hoxa\/}-4, {\it hoxb\/}-4 and {\it hoxd\/}-4

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In Drosophila, where these genes are known as HOM-C (Homeotic-complex) genes and where they have been most extensively studied, they are expressed in restricted domains along the anteroposterior axis with different anterior limits. Genetic analysis of a large number of gain- and loss-of-function alleles of these genes has revealed that these genes are important in specifying segmental identity at their anterior limits of expression. Furthermore, there is a functional dominance of posterior genes over anterior genes, such that posterior genes can dominantly specify their developmental programs in spite of the expression of more anterior genes in the same segment. In the mouse, there are four clusters of HOM-C genes, called Hox genes. Thus, there may be up to four genes, called paralogs, that are more highly homologous to each other and to their Drosophila homolog than they are to the other mouse Hox genes. The single mutants for two paralogous genes, hoxa-4 and hoxd-4, presented in this dissertation, are similar to several other mouse Hox mutants in that they show partial, incompletely penetrant homeotic transformations of vertebrae at their anterior limit of expression. These mutants were then bred with hoxb-4 mutants (Ramirez-Solis, et al. 1993) to generate the three possible double mutant combinations as well as the triple mutant. The skeletal phenotypes of these group 4 Hox compound mutants displayed clear alterations in regional identity, such that a nearly complete transformation towards the morphology of the first cervical vertebra occurs. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression. ^

Regulation of XMyoDa transcription by the binding of XMEF2A to the TATA box

MEF2 is a $\underline{\rm m}$yocyte-specific $\underline{\rm e}$nhancer-binding $\underline{\rm f}$actor that binds a conserved DNA sequence, CTA(A/T)$\sb4$TAG. A MEF2 binding site in the XMyoDa promoter overlaps with the TATA box and is required for muscle specific expression. To examine the potential role of MEF2 in the regulation of MyoD transcription during early development, the appearance of MEF2 binding activity in developing Xenopus embryos was analyzed with the electrophoretic mobility shift assay. Two genes were isolated from a X. Laevis stage 24 cDNA library that encode factors that bind the XMyoDa TFIID/MEF2 site. Both genes are highly homologous to each other, belong to the MADS ($\underline{\rm M}$CM1-$\underline{\rm A}$rg80-agamous-$\underline{\rm d}$eficiens-$\underline{\rm S}$RF) protein family, and most highly related to the mammalian MEF2A gene, hence they are designated as XMEF2A1 and XMEF2A2. Proteins encoded by both cDNAs form specific complexes with the MEF2 binding site and show the same binding specificity as the endogenous MEF2 binding activity. XMEF2A transcripts accumulate preferentially in developing somites after the appearance of XMyoD transcripts. XMEF2 protein begins to accumulate in somites at tailbud stages. Transcriptional activation of XMyoD promoter by XMEF2A required only the MADS box and MEF2-specific domain when XMEF2A is bound at the TATA box. However, a different downstream transactivation domain was required when XMEF2A activates transcription through binding to multiple upstream sites. These results suggest that different activation mechanisms are involved, depending on where the factor is bound. Mutations in several basic amino acid clusters in the MADS box inhibit DNA binding suggesting these amino acids are essential for DNA binding. Mutation of Thr-20 and Ser-36 to the negatively charged amino acid residue, aspartic acid, abolish DNA binding. XMEF2A activity may be regulated by phosphorylation of these amino acids. A dominant negative mutant was made by mutating one of the basic amino acid clusters and deleting the downstream transactivation domain. In vivo roles of MEF2 in the regulation of MyoD transcription were investigated by overexpression of wild type MEF2 and dominant negative mutant of XMEF2A in animal caps and assaying for the effects on the level of expression of MyoD genes. Overexpression of MEF2 activates the transcription of endogenous MyoD gene family while expression of a dominant negative mutant reduces the level of transcription of XMRF4 and myogenin genes. These results suggest that MEF2 is downstream of MyoD and Myf5 and that MEF2 is involved in maintaining and amplifying expression of MyoD and Myf5. MEF2 is upstream of MRF4 and myogenin and plays a role in activating their expression. ^

Swiss Shock: Minaret Rejection, European Values, and the Challenge of Tolerant neutrality

In 2009 Switzerland, for long an apparent beacon of European toleration and neutrality, voted to ban the erection of minarets. Internal religious matters are normally dealt with at the regional or local level – not at the level of the Swiss national parliament, although the state does seek to ensure good order and peaceful relations between different faith communities. Indeed, the freedom of these communities to believe and function publicly is enshrined in law. However, as a matter of national policy, now constitutionally embedded, one religious group, the Muslim group, is not permitted to build their distinctive religious edifice, the minaret. Switzerland may have joined the rest of Europe with respect to engaging the challenge of Islamic presence to European identity and values, but the rejection of a symbol of the presence of one faith – in this case, Islamic – by a society that is otherwise predominantly secular, pluralist, and of Christian heritage, poses significant concerns. How and why did this happen? What are the implications? This paper will discuss some of the issues involved, concluding the ban is by no means irreversible. Tolerant neutrality may yet again be a leitmotif of Swiss culture and not just of foreign policy.

Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Biochemical characterization of PICK1, a PKC binding protein

Protein kinase C (PKC) is a family of serine-threonine kinases that are activated by a wide variety of hormones, neurotransmitters and growth factors. A single cell type contains multiple isoforms that are translocated to distinct and different subcellular sites upon mitogenic stimulus. Many different cellular responses are attributed to PKC activity though relatively few substrates or binding proteins have been definitively characterized. We used the hinge and catalytic domain of PKC$\alpha$ (PKC7) in a yeast two-hybrid screen to clone proteins that interact with C-kinase (PICKs). One protein which we have termed PICK1 may be involved in PKC$\alpha$-specific function at the level of the nuclear membrane after activation. Binding of PICK1 to PKC$\alpha$ has been shown to be isoform specific as it does not bind to PKC$\beta$II or PKC$\alpha$ in the yeast two-hybrid system. PICK1 mRNA expression level is highest in testis and brain with lower levels of expression in skeletal muscle, heart, kidney, lung and liver. PICK1 protein contains five PKC consensus phosphorylation sites and serves as an in vitro substrate for PKC. The PICK1 protein also contains a P-Loop motif that has been shown to bind ATP or GTP in the Ras family of oncoproteins as well as the G-Protein family. Proteins which bind ATP or GTP using this motif all have some sort of catalytic function although none has been identified for PICK1 as yet. PICK1 contains a DHR/GLGF motif at the N-terminus of the protein. The DHR/GLGF motif is contained in a number of recently described proteins and has been shown to mediate protein-protein interactions at the level of membranes and cytoskeleton. When both PKC$\alpha$ and PICK1 are co-expressed in Cos1 cells the two proteins co-localize to the perinucleus in immunoflouresence studies and co-immunoprecipitate. The binding site for PKC7 has been localized to amino acids 1-358 on PICK1 which contains the DHR/GLGF motif. Binding of PICK1 to PKC$\alpha$ requires the hinge and C-terminal domains of PKC$\alpha$. In vitro, PICK1 binds to PKC$\alpha$ and inhibits its activity as assayed by myelin basic protein phosphorylation. PICK1 also binds to TIS21, a primary response gene that is expressed in response to phorbol ester and growth factor treatment. The Caenorhabditis elegans homologue of PICK1 has been cloned and sequenced revealing a high degree of conservation in the DHR/GLGF motif. A more C-terminal region also shows a high degree of conservation, and the C. elegans PICK1 homologue binds to PKC7 suggesting a conservation of function. Taken together these results suggest that PICK1 may be involved in a PKC$\alpha$-specific function at the level of the nuclear membrane. ^