Research Interests of Dr. George Molloy, Associate Professor (revised 2-15-00)

Discipline: Molecular and developmental biology, genetics and neurobiology.

Current interest is in the molecular mechanisms which: i) control (possible) tissue-specific transcription of the rat brain creatine kinase (CKB) gene in neural and glial cells and ii) control transcription of CKB mRNA during the development of glial cells in the oligodendrocyte lineage and during embryonic development iii) the physiological role of the CKB protein in differentiated astrocytes and oligodendrocytes. This is important since creatine kinase (CK) is a metabolic enzyme involved in maintenance of high ATP levels in cells where energy requirements are high (e.g. glial and neural cells). Knowledge of how the cell controls the transcription of CKB mRNA is important since it will help explain how the cell controls transcription of genes involved in control of growth of normal cells as well as genes which are activated during the differentiation of glial cells (i.e. astrocytes and oligodendrocytes).

CKB mRNA transcription is under (possible) tissue-specific regulation being highly transcribed in the brain but repressed in a number of other tissues. Therefore, we are interested in defining the cis-acting CKB promoter sequences and trans-acting nuclear factors regulating CKB transcription.

Studies on the expression of rat CKB represent an attractive experimental system because:

1. The CKB gene has been cloned and sequenced and the ezymatic function of the CKB enzyme is well understood - CKB serves to regenerate ATP in cell types which expend ATP quickly and/or in large amounts.

2. CKB is expressed in highest levels in the brain and our recent work has shown CKB is highest in brain glial cells (i.e. astrocytes and oligodendrocytes).

3. The expression and function of CKB during the differentiation of brain astrocytes and oligodendrocytes can be studied since astrocytes and oligodendrocytes and their precursors can be isolated from rat brain and their differentiation can be followed relatively easily.

4. CKB is relevant to a number of disease situations:

Current research emphasizes:

1. CKB gene cloning and in vivo expression of the CKB gene following gene transfer (i.e. DNA transfection) into either exponentially-growing or differentiating

rat glial (e.g. C6 glioma) and neural cells.

2. Identification of cis-acting regulatory DNA sequences in the CKB promoter and far-upstream regulatory elements (URE) which influence CKB mRNA transcription and isolation of the trans-acting nuclear proteins which interact with these regulatory sequences.

3. Site-directed mutagenesis of the CKB promoter TATA and CCAAT box regions as well as the CKB transcription start site region (from +1 to +5 bp) to determine the important regulatory sequences.

4. Identification of the repressor-like protein in mouse fibroblast cells which

binds to the CKB promoter and prevents transcription in NIH 3T3 fibroblasts.

5. Investigating the timing of expression of CKB during the differentiation rat rat brain astrocytes and oligodendrocytes in cell culture.

6. Understanding the molecular mechanism by which cyclic AMP and the CREB protein increases transcription of the CKB gene.

7. Of more future interest, determining which regions in the brain express high levels of CKB mRNA and CKB enzyme by using in situ hybridization techniques and investigating CKB sequence elements which may regulate possible tissue-specific CKB mRNA expression by introducing mutated CKB genes into transgenic mice.

Research Interests of Dr. George Molloy (revised 2-15-00)

Introduction:

Listing of current projects page 3

Background and Discussion of individual projects page 5

References and manuscripts in preparation page 10

We are interested in the (i) regulation of transcription of the rat brain creatine kinase (CKB) mRNA and the (ii) physiological role of the CKB protein in the cells which compose the central nervous system (CNS) and the peripheral nervous system (PNS). We have shown in primary cultures from rat brain that CKB mRNA levels are much higher in astrocytes and oligodendrocytes than in neurons (J. Neurochemistry 59: 1925-1932 [1992]) and we have suggested that high expression of CKB protein facilitates the energy-demanding events during myelinogenesis in oligodendrocytes and ion transport in astrocytes. Also, it is likely that CKB mRNA and protein are high in Schwann cells, which carry out the myelination of neurons in the PNS; this is also under investigation (in preparation).

The creatine kinase (CK) (EC 2.7.3.2) isoenzymes are important in some cell types, since they catalyze the reversible transfer of a high-energy phosphate group from creatine phophate (CrP) to ADP to regenerate ATP.

The brain CK (CKB) and muscle CK (CKM) are cytoplasmic enzymes that are catalytically active as dimers and exist in three isozymic forms: MM (predominant in adult skeletal muscle), BB (predominant in brain) and MB (predominant in cardiac muscle). The mitochondrial CK (CKmi) self-associates to form an octamer and is located on the outer surface of the inner mitochondrial membrane. To explain the requirement for the different CK isoforms, a CK "energy shuttle" has been proposed wherein the CKmi uses the ATP produced by oxidative phosphorylation to generate creatine phosphate (CrP), a high-energy phosphate molecule, which is subsequently transported to the cytoplasm and used by the cytoplasmic form of CK (either CKB or CKM) to regenerate ATP at sites of high ATP consumption.

The CKM gene is expressed in a highly tissue-specific manner being expressed almost exclusively in skeletal and cardiac muscle; however, CKM recently it has also been detected at very low levels in discrete regions of the brain (e.g. the Perkinge cells). Conversely, CKB is expressed in a broader range of tissues and is high in brain, lower in stomach and heart and barely-detectable in liver. While the functional role of the CKB protein in the brain is not fully understood, it has been localized to brain synaptic plasma membranes possibly coupled to Na/K-ATPase and to acetylcholine receptor-rich membranes. This suggests CKB may regenerate the ATP necessary for transport of ions and neurotransmitters. We have recently shown that the cell type in the brain with the highest CKB mRNA expression are the glial cells: primary cell cultures of astrocytes and oligodendrocytes contained 15- to 17-fold higher levels of CKB mRNA than primary neuronal cells (Molloy et al, 1992). High CKB mRNA expression was also observed in established C6 glioma cells (Wilson et al., 1993) and we are currently using C6 glioma as a model system for some aspects of the research.

Since some aspects of the above goals are more easily conducted with established cell lines, we have shown that expression of CKB mRNA and protein is high in rat C6 glioma (which is a glioblastoma from the CNS; J. Neuroscience Res. 35: 92-102 [1993]) and rat RT4-D6 peripheral glial neurotumor cells (which have many characteristics of Schwann cells; Wilson et al. [1995] Dev. Biol. [submitted]).

In the CNS, astrocytes and oligodendrocytes arise from bipotential progenitor cells (called "02A" cells), which can differentiate into either oligodendrocytes or type 2 astrocytes depending on the inducing molecules in the extracellular environment (see Louis et al. J. Neuroscience Res. 31: 193-204 [1992]). Indeed, Raible and McMorris (J. Neuroscience Res. 27: 43-46 [1990]) have shown that the differentiation of primary 02A progenitor cells into oligodendrocytes in cell culture is stimulated by the elevation of cAMP. Since it is likely that CKB transcription is activated during the differentiation of oligodendrocytes and astrocytes, we have recently investigated whether there is an increase in CKB mRNA transcription during the following events: (i) in cells where cAMP has been elevated and (ii) during the differentiation of (established) rat brain CG4 cells, which are characteristic of 02A progenitor cells and can be triggered to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium (see below).

With regard to the role of cAMP and CKB expression, we have recently shown in established U87 human glioblastoma cells that CKB mRNA transcription is increased when cAMP levels are elevated by the addition of either (1) forskolin, which activates the adenylate cyclase enzyme; (2) prostaglandin E1 (PGE1) or PGE2, which are ligands found in the brain that bind to their G-protein-coupled membrane receptor and activate the Ga s protein causing increased synthesis of cAMP; or cholera toxin (CTX) (Kuzhikandathil and Molloy, 1994; 1995). The stimulation by CTX (which directly activates the Ga s protein and leads to the activation of adenylate cyclase) is extremely important since it suggests that PGE1 and PGE2 may be physiological ligands which activate CKB mRNA transcription by interacting with their G-protein-coupled receptor; this possibly may also occur during the differentiation of 02A progenitor cells. Therefore, these findings give rise to Project 1 described below:

Project 1: Determine with primary cultures of purified (immature) oligodendrocytes and astrocytes from day 2 neonatal rats (i) if elevating the level of cAMP (with either forskolin, cholera toxin, PGE1 or PGE2) activates CKB transcription and, if so, (ii) identify which cis-acting CKB gene element(s) and what nuclear DNA-binding protein(s) are important for this activation.

Project 2: Determine with primary cultures of purified oligodendrocytes and astrocytes from day 2 neonatal rats what percentage of the CKB enzyme is located in the cytoplasm and the nucleus of the cell and how this may change as these glial cells (i) further mature and differentiate in cell culture and/or (ii) have their level of cAMP increased by forskolin. This may also be determined in conjunction with Project 3, where primary oligodendrocytes and astrocyte cultures will be prepared at weekly intervals during the first six weeks after birth; here, glial cell differentiation will occur in vivo.

Project 3: Determine if the 5-fold increase in CKB enzyme activity observed during the period of postnatal day 1 to day 40 is due to either an increase in CKB mRNA (possibly resulting from activated CKB transcription) or simply the result of posttranslational protein modification which increases CKB enzyme activity.

Project 4: Determine if CKB transcription is regulated in a "negative feedback-loop" manner which is controlled by the level of active CKB enzyme in the cell. Here, we will use the creatine analogs b -GPA and cyclocreatine to inhibit CKB enzyme activity in U87 glioblastoma and C6 glioma cells and measure the effect on CKB transcription.

Project 5: Determine (i) if CKB transcription is activated sometime during the differentiation program of 02A progenitor cells as they give rise to either oligodendrocytes and/or astrocytes and, if so, (ii) identify which cis-acting CKB gene element(s) and what nuclear DNA-binding protein(s) are important for this activation and (iii) whether cAMP is involved in this activation of CKB transcription.

Project 6: Determine if the high expression of CKB enzyme activity observed in glial cells is necessary for myelinogenesis in oligodendrocytes and ion transport in astrocytes?

Project 7: Measure CKB mRNA levels in various tissues during embryonic development in the rat. This will establish the timing of CKB expression in different embryonic tissues and also compare CKB mRNA levels in embryonic brain with CKB mRNA in other embryonic tissues and organs. This would provide the framework to identify the CKB gene regulatory DNA elements responsible for expression.

Project 8: Block CKB expression during (i) embryonic development and (ii) the early postnatal period in the rat, using the inducible expression of anti-sense CKB RNA under the control of the tetracycline (Tet) promoter system to interfere with the expression of CKB mRNA.

Project 9: We have also found recently that CKB expression is almost undetectable in mouse NIH3T3 and Balb/c 3T3 fibroblasts and that the sequences at the transcription initiation region (i.e. the INR) in the CKB gene (from -5 bp to +5 bp: [-5]CGGCCGTCGT[+5]) mediate this repression of the rat CKB gene when it is transfected into NIH3T3 fibroblasts. This suggests that the CKB INR is acting as a negative cis-acting element for a trans-acting repressor protein. (Parameswaran et al., 1995a). Conversely, in HeLa cells, the INR increases CKB expression about 5-fold suggesting the INR is acting as a positive cis-acting element for a trans-acting activator protein.

We would like (i) to identify and characterize the potential repressor protein in mouse fibroblasts which binds to the CKB INR element and understand how it depresses CKB transcription and (ii) to determine if the CKB INR element also participates in the low CKB expression we have observed in neuronal cell lines and possibly in cultured primary neuronal cells and (iii) to ask if the potential repressor protein in mouse fibroblasts also functions to depress CKB in neuronal cells.

Project 10: Can the CKB gene be activated by cotrasfection of cDNA for the recently cloned MEF2C protein which binds to sequences similar to the -60 TATAAATA in the CKB promoter; MEF2C may be the same or similar to the TARP protein initially identified by Hobson et al. (1988) ? If so, what is the mechanism by which MEF2C acts? Does MEF2C simply act as a positive regulator or does it also prevent repression?

Project 11: Determine if the depression of CKB expression in NIH 3T3 fibroblasts can be reversed by cotransfection of the cDNA expressing the recently-characterized YY-1 protein which binds to sequences similar to the [-5]CGGCCGTCGT[+5] in the CKB INR and has been shown to either activate or repress gene expression depending cell culture conditions.

Project 1: While we have shown that an elevation of cAMP will increase CKB transcription in U87 glioblastoma cells, it must be established that cAMP activates CKB transcription in primary cultures of glial cells and that this activation is not simply an artifact of the established U87 cells.

Therefore, primary cultures of purified oligodendrocytes and astrocytes, derived from 1-day-old rats, will be treated for about 12 hrs with either forskolin or PGE1 or PGE2 or CTX to elevate cAMP. Total cellular RNA will be assayed for CKB mRNA using the rat CKB Exon 7,8 probe in the RNase-protection assay. We have shown this probe detects fully-spliced exon 7 and exon 8 RNA in mature CKB mRNA as well as the unspliced CKB mRNA precursor, which is one indicator of increased CKB transcription. Since it may be difficult to isolate sufficient quantities of primary oligodendrocytes and primary astrocytes to obtain from 2 to 10 m g of RNA, we may have to increase the sensitivity of our normal RNase-protection assay by:

Project 2: Manos and Bryan have recently measured the distribution of CKB enzyme activity in the nucleus and cytoplasm of primary oligodendrocytes and astrocytes in cell culture derived from day 2 neonatal rats (Dev. Neurosci. 15: 271-279 [1993]). In oligodendrocytes, CKB enzyme was present in both the nucleus and cytoplasm and was colocalized with MBP, indicating CKB expression was high when synthesis of MBP was high. This analysis was not carried further because oligodendrocytes were considered to be "asynchronous and heterogeneous with respect to developmental stage and cell division".

However, the cellular distribution of CKB enzyme was examined further in primary astrocytes (at both subconfluent and quiescent contact-inhibited cell densities), since astrocytes were found to be relatively "homogeneous with respect to cell division at confluence". In subconfluent astrocytes, immunofluorescent-antibody staining showed intense CKB staining in both the nucleus and cytoplasm and about 92% of the nuclei were intensely stained. In confluent astrocytes, however, there was a significant reduction in the CKB staining in the nucleus since only about 47% of the nuclei were intensely stained. In agreement, cell fractionation studies showed that the CKB enzyme activity in nuclei from subconfluent astrocytes was about 3-fold higher than in nuclei from confluent astrocytes. However, in both subconfluent and confluent astrocytes, the majority (60 to 80%, respectively) of the CKB enzyme activity was present in the cytoplasm. These authors cite three amino acid regions in the CKB protein which resemble a "nuclear localization sequence" (i.e. residues 10-14; 129-137 and 312-319).

We wish to determine the subcellular location of the CKB protein in astrocytes and oligodendrocytes as they further mature and differentiate.

Therefore, we will determine with primary cultures of purified oligodendrocytes and astrocytes from day 2 neonatal rats what percentage of the CKB enzyme is located in the cytoplasm and the nucleus of the cell and how this may change as these glial cells (i) further mature and differentiate in cell culture and/or (ii) have their level of cAMP increased by forskolin. This may also be determined in conjunction with Project 3, where primary oligodendrocytes and astrocyte cultures will be prepared at weekly intervals during the first six weeks after birth. Here, of course, the glial cell differentiation will be occurring in vivo in the brain, prior to the preparation of the primary cell cultures.

Project 3: Manos et al. have shown that CKB enzyme activity per m g of protein in total rat brain increases about 5-fold during the period of postnatal day 1 to day 40 and more than half of the CKB activity was present in oligodendrocytes (J. Neurochemistry 56:2101-2107 [1991]). We would like to determine if this is due to either an increase in CKB mRNA, possibly resulting from activated transcription, or simply the result of posttranslational protein modification which increases CKB enzyme activity.

For this goal, we would prepare primary cultures of oligodendrocytes from postnatal rat brain at 5 day intervals between day 1 and day 40 and measure the CKB mRNA levels with the rat CKB Exon 7,8 probe. As mentioned above, since the CKB Exon 7,8 probe will detect increased CKB pre-mRNA resulting from increased transcription, any observed increase in CKB pre-mRNA in the oligodendrocytes during this postnatal period would establish increased CKB transcription.

Project 4: Determine if CKB transcription is regulated in a "negative feedback-loop" manner which is controlled by the level of active CKB enzyme in the cell. Here, we will use the creatine analogs b -GPA and cyclocreatine to inhibit CKB enzyme activity in U87 glioblastoma and C6 glioma cells and measure the effect on CKB transcription.

Project 5: It has been shown previously that an elevation in cAMP will (i) accelerate the differentiation of primary 02A progenitor cells into oligodendrocytes in cell culture (Raible and McMorris [1990] JNR 27: 43-46) and (ii) activate CKB transcription in U87 glioblastoma (Kuzhikandathil and Molloy, 1994; 1995). Therefore, it is important to determine if CKB transcription is activated sometime during the differentiation program of 02A progenitor cells as they give rise to either oligodendrocytes and/or astrocytes.

Our preliminary experiments agree with the above, since we have recently shown using Northern blot analysis that CKB mRNA levels are significantly increased when (undifferentiated) CG4 progenitor cells are stimulated to differentiate into either oligodendrocytes or astrocytes. Therefore, the following points are important to Project 5:

Project 6: Is the high expression of CKB enzyme activity observed in glial cells necessary for myelinogenesis in oligodendrocytes and for ion transport in astrocytes? To address this question, we will determine:

(ii) We will block the production of the CKB enzyme in myelinating glial cells by employing the controlled synthesis of CKB anti-sense RNA expressed from the CKB cDNA cloned under the control of the inducible Tetracycline (Tet) promoter. This approach using CKB anti-sense RNA is likely to be the most specific since it should only affect CKB mRNA; in addition, the synthesis of CKB anti-sense RNA can be quickly induced to very high levels and rapidly reversed. Note that in Project 5, CKB anti-sense RNA will also be used in attempts to block the rapid increase in CKB enzyme activity which normally occurs between postnatal days P1 and P30.

(2) Whether during the time course of differentiation and myelination of CG4 and/or peripheral RT4-D6 glial cell, the increase in CKB mRNA and protein occurs prior to myelinogenesis, as would be expected if CKB was necessary for myelination.

Project 7: We have currently measured the level of CKB mRNA in 17 discrete regions of the adult rat brain, using the rat CKB Exon 7,8 probe. However, because CKB mRNA is expressed in a number of different tissues, it is of interest to measure CKB mRNA levels in various tissues during embryonic development. This would not only reveal the timing of expression in the different regions of the embryonic brain but would permit a comparison of the

CKB mRNA levels in embryonic brain with CKB mRNA in other embryonic tissues and organs. This would provide the framework to identify the CKB gene regulatory DNA elements responsible for expression. For example, CKB enzyme levels are high in the liver at birth but dramatically decrease shortly after birth (Manos et al. J. Neurochemistry 56: 2101-2107 [1991]). Therefore, it is possible that CKB transcription is high in embryonic liver and is repressed after birth. Two approaches would be used toward this goal.

(1) A nonradioactive anti-sense CKB RNA probe labelled with digoxygenin would be used for in situ hybridization of "whole-mount" mouse (or rat) embryos at appropriate times during development; this would establish which tissues express high or low CKM mRNA.

(2) The CKB gene regions responsible for this pattern of expression would then be examined. Results published thus far on CKB leave open the possibility that regulatory elements could be situated either (i) in the far-upstream or proximal promoter; (ii) within the introns; or (iii) within the 3' UTS or 3' flanking sequence. Therefore, to detect any of these possibilities, the b -lac Z (reporter) gene will be cloned in frame into the 3' end of the CKB coding region in exon 8 to create a hybrid CKB-b -gal fusion gene. Thus, examining the expression of the CKB-b -gal gene would detect any potential regulatory elements located within introns, or the 3'UTS or the 5' and 3' flanking sequences. This hybrid gene will be introduced into fertilized mouse eggs which then will be implanted into pregnant mothers and the developmental expression can easily be assayed using the convenient "blue color assay" for b -gal either in whole-mount embryos or tissue slices. Initially, we will compare the (i) magnitude and (ii) tissue-specific expression of the CKB-b -gal gene under the control of the native long promoter (2.9 kb) versus the short (0.2 kb) promoter to test for upstream regulatory elements (UREs). Later, we will examine if regulatory elements are located in any of the introns, the 3'UTS or 3' flanking sequence. The blue color assay will permit the rapid assay for CKB gene elements regulating expression either during (i) embryonic development or (ii) the early postnatal period of increased CKB expression.

Project 8: Here, we will attempt to block CKB expression during (i) embryonic development and (ii) the early postnatal period, using the inducible expression of anti-sense CKB RNA under the control of the Tet promoter system to interfere with the expression of CKB mRNA. In these experiments, Tet promoter system will NOT express anti-sense CKB RNA in the presence of Tetracycline but only in its absence. This system works well not only in tissue culture (where tetracycline is simply added to the medium) but in whole animal systems as well (where tetracycline pellets are implanted subcutaneously); in addition, there are now forms of tetracycline which effectively cross the blood-brain barrier.

Project 9: CKB has a potentially complex promoter containing a consensus TATAAA at -60 bp and a nonconsensus TTAA at -25 bp plus CCAAT boxes at -55 bp and -85 bp. In all cell types examined thus far, however, initiation of in vivo transcription was directed only from the -25 TTAA (Hobson et al., 1988; 1990).

We have also found recently that CKB expression is almost undetectable in mouse NIH3T3 and Balb/c 3T3 fibroblasts and that the sequences at the transcription initiation region (i.e. the INR) in the CKB gene (from -5 bp to +5 bp: [-5]CGGCCGTCGT[+5] mediate repression of the rat CKB gene when it is transfected into NIH3T3 fibroblasts, suggesting the INR is acting as a negative cis-acting element for a trans-acting repressor protein. (Parameswaran et al., 1995). Conversely, in HeLa cells, the INR increases CKB expression about 5-fold suggesting the INR is acting as a positive cis-acting element for a trans-acting activator protein.

We would like (i) to identify and characterize the potential repressor protein in mouse fibroblasts which binds to the CKB INR element and understand how it depresses CKB transcription and (ii) to determine if the CKB INR element also participates in the low CKB expression we have observed in neuronal cell lines and possibly in cultured primary neuronal cells and (iii) to ask if the potential repressor protein in mouse fibroblasts also functions to depress CKB in neuronal cells.

Project 10: Can the CKB gene be activated by cotrasfection of cDNA for the recently cloned MEF2C protein which binds to sequences similar to the -60 TATAAATA in the CKB promoter; MEF2C may be the same or similar to the TARP protein initially identified by Hobson et al. (1988) ? If so, what is the mechanism by which MEF2C acts? Does MEF2C simply act as a positive regulator or does it also prevent repression?

Project 11: Determine if the depression of CKB expression in NIH 3T3 fibroblasts can be reversed by cotransfection of the cDNA expressing the recently-characterized YY-1 protein which binds to sequences similar to the [-5]CGGCCGTCGT[+5] in the CKB INR and has been shown to either activate or repress gene expression depending cell culture conditions.

Recent publications: (revised 2-15-00)

Mukherjee, R. and Molloy, G.R. (1987). DRB inhibits transcription of the ß-hemoglobin gene in vivo at initiation. J. Biol. Chem. 262, 13697-13705.

Hobson, G.M., Mitchell, M.T., MOLLOY, G.R., Pearson, M.L. and Benfield, P.A. (1988) Identification of a novel TA-rich DNA binding protein that recognizes a TATA sequence within the brain creatine kinase promoter. Nucleic Acids Research 16: 8925-8944.

Hobson, G.M., MOLLOY, G.R., and Benfield, P.A. (1990) Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene. Mol. Cell. Biol. 10: 6533-6543.

MOLLOY, G.R., C.D. Wilson, P. Benfield, J. deVellis and S. Kumar (1992) Expression of the rat brain creatine kinase gene is high in primary astrocytes and oligodendrocytes and low in neurons. J. Neurochemistry 59: 1925-1932.

Wilson, C.D., Parameswaran, B. and MOLLOY, G.R. (1993) Expression of the brain creatine kinase gene in rat C6 glial cells. J. Neuroscience Research 35: 92-102.

Kuzikanithil, E., and MOLLOY, G.R. (1994) Transcription of the rat brain creatine kinase gene in glial cells is modulated by cAMP-dependent protein kinase. J. Neuroscience Res. 39: 70-82.

Zhao, J., Schmieg, F., Simmons, D.T. and MOLLOY, G.R. (1994) Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene. Mol. Cell. Biol. 14: 8483-8492.

Kuzhikandathil, E., and MOLLOY, G.R. (1995) Prostaglandin E₁ and E₂ and Cholera toxin increase transcription of the rat brain creatine kinase gene in the human U87 glioblastoma cell line. GLIA 15: 471-479.

Ilyin, S.E., Plata-Salamon, C.R., MOLLOY, G.R., Sonti, G. (1996) Creatine Kinase B mRNA levels in brain regions from male and female rats. Molecular Brain Research 41: 50-56.

Zhao, J., Schmieg, F., Dodgson, N., Simmons, D.T. and MOLLOY, G.R. (1996) p53 binds to a novel recognition sequence in the proximal promoter of the rat muscle creatine kinase gene and activates its transcription. ONCOGENE 13:293-302.

Wilson, C.D., Shen, W., Kuzhikandathil, E. and G.R. Molloy (1997) Expression of the brain creatine kinase gene in rat RT4 neurotumor cells and its modulation by cell confluence. Developmental Neuroscience 19: 384-394.

Wilson, C.D., Shen, W., and G.R. Molloy (1997) Expression of the brain creatine kinase gene is low in neuroblastoma cells. Developmental Neuroscience 19: 375-383.

Kuzhikandathil. E. and MOLLOY, G.R. (1999) The proximal promoter of the rat brain creatine kinase gene lacks a concensus CRE element but is essential for the cAMP-mediated increased transcription in glioblastoma cells. J. Neroscience Res. 56: 371-385.

Parameswaran, B., Shen, W. and MOLLOY, G.R. (1999) Conditions providing enhanced transfection efficiency permit analysis of the far-upstream and proximal promoter of the rat brain creatine kinase gene in rat pheochromocytoma PC12 cells. J. Neuroscience Methods 92: 3-13.

MANUSCRIPTS IN PREPARATION (writing is in progress but all experiements have ben completed)

(1a) Shen, W., Antolik, C. and Molloy, G.R. (2000) Expression of the brain creatine kinase gene during postnatal development of rat brain cerebellum. J. Neroscience Res. (submitted).

(1b) Shen, W., Antolik, C. and Molloy, G.R. (2000) Expression of the brain creatine kinase gene during postnatal development of rat brain cerebrum. J. Neroscience Res. (submitted).

(1c) Shen, W., and Molloy, G.R. (2000) Expression of the brain creatine kinase gene increases during differentiation of rat brain cerebral oligodendrocytes. J. Neroscience Res. (submitted).

(2) Kuzikanithil, E., Ilyin, S.I., Zhao, J. and MOLLOY, G.R. (2000) Cell-line specific transcriptional and posttranscriptional regulation of the rat brain creatine kinase gene by introns. J. Biol. Chem. (in preparation)

(3) Wilson,C.D. and G.R. MOLLOY (2000) Expression of the brain creatine kinase gene is induced by sodium butyrate. J. Biol. Chem. (in preparation).

(4) Parameswaran, B., C.D. Wilson and G.R. MOLLOY. (2000) Negative regulation of the rat brain creatine kinase gene by sequences at the site of transcription initiation. J. Biol. Chem. (in preparation).

(5) Parameswaran, B., Wilson, C.D., Megee, P., and MOLLOY, G.R. (2000) Expression of the brain creatine kinase gene in rat pheochromocytoma PC 12 cells. J. Neurochemistry (in preparation)

(6) Parameswaran, B., Wilson, C.D., Megee, P., Kim, L. and MOLLOY, G.R. (2000) Expression of the brain creatine kinase gene in parental rat pheochromocytoma PC 12 cells and cloned stable variants. J. Biol. Chem. (in preparation).

(7) Parameswaran, B., C.D. Wilson, Zhao, J. and G.R. MOLLOY. (2000) Negative regulation of the rat brain creatine kinase gene by far-upstream 5' elements. J. Biol. Chem. (in preparation).

(8) Mukherjee, R. and MOLLOY, G.R. (2000) Transcription of the ß-major hemoglobin gene ceases in terminally differentiated murine erythroleukemic cells. J. Biol. Chem. (in preparation)