A Novel Family of Cys-Cys, His-Cys Zinc Finger Transcription Factors Expressed in Developing Nervous System and Pituitary Gland*

A screen designed to identify proteins that specifi- cally bind to retinoic acid response elements resulted in the identification of a rat cDNA encoding a novel pro- tein containing six Cys-Cys, His-Cys zinc fingers. This gene is expressed in a restricted fashion exhibiting dis- tinct temporal and spatial patterns in the developing nervous system, primarily brain, spinal cord, sensory ganglia, retina, and nasal epithelia, as well as in the pituitary, and is referred to as neural zinc finger factor 1 (NZF-1). NZF-1 binds specifically to a cis -regulatory element of the (cid:98) -retinoic acid receptor (RAR (cid:98) ) gene, as well as to other related DNA elements, including two in the upstream enhancer region of the mouse Pit-1 gene. In heterologous cells, NZF-1 activates transcription from promoters containing specific binding sequences and can synergize with other factors, such as Pit-1, to regulate gene expression. These results suggest that NZF-1 may exert regulatory roles in the developing and mature nervous system and in the pituitary gland. Identification of a second mouse gene highly homologous to NZF-1, encoded by a distinct genomic locus, reveals a dispersed gene family encoding proteins containing

Precise temporal and spatial patterns of development are controlled by sequential activation of a hierarchy of regulatory genes, which encode transcription factors containing multiple classes of DNA binding motifs. Zinc coordinated fingers are one of the most common DNA binding motifs among eukaryotic transcription factors and are classified based on amino acid sequence of the zinc fingers. The Cys-Cys, His-His class, which is typified by the Xenopus transcription factor IIIA (1), contains the largest number of members. These proteins contain two or more fingers in a tandem repeat. In contrast, steroid receptors, such as the glucocorticoid receptor, contain only two zinc coordinated structures with four (C 4 ) and five (C 5 ) conserved cysteines. The third class of zinc fingers, which also binds to single-stranded nucleic acids, has a consensus sequence of Cys-X 2 -Cys-X 4 -His-X 4 -Cys. Such factors are found in transposable element copia, plants, and mammalian cells as well as in retroviruses. Other metal-coordinating proteins have different structures such as C 6 in the yeast GAL4 protein and a cysteinerich structure in the E1A oncoprotein (2).
In accordance with their structural diversity, zinc finger proteins play a variety of important roles in cell growth, differentiation, and development. Transcription factor IIIA and the ubiquitous transcription factor SP1 are broadly involved in the regulation of transcription, whereas the Drosophila zinc finger proteins Krü ppel and Hunchback are crucial for proper segmentation of the developing embryo (3)(4)(5). In humans, mutations in a kidney zinc finger protein (WT1) result in Wilm's tumor (6,7). Recently, a zinc finger protein (REST) has been shown to repress neuronal gene expression in non-neuronal tissues (8,9).
Because retinoic acid receptor (RAR) 1 binds ineffectively to DNA response elements, requesting a DNA binding co-regulator, expression screening was performed with a ␤-retinoic acid response element (␤RARE). This proved to be a successful strategy for the cloning of a retinoic acid receptor co-regulator which was identified as a member of the retinoid X receptor family (10 -12). This co-regulator binds DNA cooperatively as a heterodimer with the retinoic acid receptor and other nuclear receptors (11)(12)(13)(14)(15)(16).
In order to investigate the mechanisms that underlie regulation of the expression of the ␤-retinoic acid receptor (RAR␤) and Pit-1 genes further, we screened expression libraries based on the detection of protein-DNA interactions using the ␤RARE and the retinoic acid response element of the Pit-1 gene enhancer (17). In this manuscript we report that, as a consequence of these screens, we have identified a novel transcript encoding a Cys-Cys, His-Cys zinc finger protein. Based on its restricted expression pattern in the nervous system, this factor is referred to as NZF-1 (neural zinc finger factor 1). Characterization of NZF-1 has revealed the presence of two separate Cys-Cys, His-Cys type zinc finger DNA binding domains, each of which can bind independently to similar DNA sequences. The preferred consensus sequence is distinct from the consensus retinoic acid receptor response element. The NZF-1 gene exhibits a restricted pattern of expression in the nervous system, testis, and pituitary gland. NZF-1 can serve as a transcription factor and elicit synergistic activation with other factors, such as Pit-1. Our results suggest that this novel zinc finger protein may exert roles both in the development and function of the central nervous system and the anterior pituitary gland.

MATERIALS AND METHODS
Cloning of NZF-1-A concatemerized ␤RARE oligonucleotide (5Ј-AATTGGGTTCACCGAAAGTTCAC-3Ј) was used to screen a rat pituitary GC cell gt11 expression library as described previously (18). Multiple, overlapping NZF-1 clones were subsequently isolated from rat cerebellum and pituitary cell line cDNA libraries by DNA hybridization screening. cDNA inserts were subcloned into pBKSII(Ϫ) (Stratagene) and sequenced as double-stranded templates using a Sequenase 2.0 kit (U. S. Biochemical Corp.).
RNase Protection Assays and in Situ Hybridization Analysis-RNase protection assays were performed using 32 P-radiolabeled probes, as described previously (19). Hybridization reactions contained 10 g of yeast tRNA or total tissue RNA. A rat ␤-actin probe (280 nucleotides) was included as a control when indicated. Ribonucleotide probes were transcribed in vitro using constructs containing a 610-bp fragment of the NZF-1 cDNA coding for amino acids 446 -648 (probe A) or a 390nucleotide fragment coding for amino acids 753-882 (probe B) as templates.
For in situ hybridizations, cryosections of rat embryonic tissues were mounted on poly-L-lysine-coated slides and air-dried. Pretreatment, hybridizations, and washing conditions have been described (20). Briefly, sections were digested with proteinase K (10 g/ml, 37°C, 30 min), acetylated, dehydrated, and dried. 80 l of hybridization mixture containing 35 S-labeled probe (10 7 cpm/ml) was spotted on each slide. After incubation at 55°C for 16 h, slides were rinsed, digested with ribonuclease A (20 g/ml, 37°C, 40 min), and washed in 0.1 ϫ SSC for 30 min at 65°C. After dehydration, slides were dipped in Kodak NTB2 autoradiographic emulsion (diluted 1:1 with distilled water), dried in a humid chamber (2-4 h), exposed desiccated in the dark at 4°C for two weeks, and developed with Kodak D-19, followed by Autoradiographic Fixer.
Genomic Southern Blot Analysis and Mouse Chromosomal Localization-Duplicate samples (10 g) of mouse and rat genomic DNA were digested separately with restriction endonucleases BamHI, BglII, and SacI, electrophoresed through a 0.7% agarose gel, and transferred to a nylon membrane. The radiolabeled probes used were a 400-bp fragment from rat NZF-1 and a 1.2-kb fragment from mouse NZF-2. Hybridizations were carried out in 2 mM EDTA, 20 mM Tris, pH 7.6, 0.5% SDS, 6 ϫ SSC, 5 ϫ Denhardt's solution, and 100 g/ml carrier salmon sperm DNA at 65°C. After washing under high stringency conditions (0.3 ϫ SSC at 65°C), the membrane was autoradiographed.
Mapping of the genes for Nzf-1 and Nzf-2 in mouse was performed by linkage analysis of an interspecific backcross. Progeny from a (C57BL/6J ϫ Mus spretus) F1 ϫ C57BL/6J backcross were typed for about 350 genetic markers spanning the mouse genome as described previously (21). To identify restriction fragment length variants (RFLVs), DNAs from the parental strains and F1 mice were digested with 10 different restriction enzymes followed by Southern analysis. A 1060-bp XhoI-BamHI fragment from the rat NZF-1 cDNA and a 1.3-kb XbaI fragment from the mouse NZF-2 cDNA were utilized as probes. Blots were hybridized with 32 P-labeled probes at 65°C and washed in 1.0 ϫ SSC, 0.1% SDS at 50°C essentially as described previously (21).
Protein Overexpression, Electrophoretic Mobility Shift Assays, and DNA Methylation Interference Analysis-In order to study the in vitro DNA binding properties of this factor, fragments of the NZF-1 cDNA were subcloned into the pGEX-KG vector (22) and GST fusion proteins were expressed in Escherichia coli BL21 (DE3). Construct 2ZF was generated by subcloning a 990-bp NcoI-XhoI fragment of cDNA clone 13A into the corresponding sites of pGEX-KG, while 1ZF was constructed with an equivalent NcoI-XhoI fragment from clone 3B which has a deletion of 135 nucleotides encoding a zinc finger (Fig. 1B). An additional 1.4-kb XhoI-XhoI fragment encoding the C-terminal portion of NZF-1 was introduced into 2ZF to make 5ZF. 3ZF was generated by subcloning a polymerase chain reaction fragment encoding three C-terminal zinc fingers into the pGEX-KG vector.
In electrophoretic mobility shift assays, purified GST fusion proteins were preincubated in 20 l of binding buffer (50 mM HEPES, pH 7.9, 50 mM KCl, 5% glycerol, 2 mg/ml bovine serum albumin, 10 M ZnSO 4 ) containing 1 g of poly(dI-dC) and 0.1 g of salmon sperm DNA for 5 min. Samples were incubated for an additional 25 min at room temperature after adding 32 P-radiolabeled probe (0.1-0.5 ng). 4.5 l of each reaction was loaded onto a 5% nondenaturing 0.5 ϫ TBE polyacrylamide gel and electrophoresed at 20 V/cm for 60 min. Gels were dried and autoradiographed.
For methylation interference experiments, oligonucleotides were labeled at the 5Ј end using T4 kinase and ␥-[ 32 P]ATP. After annealing, double-stranded probes were gel-purified and methylated with dimethyl sulfate. Binding reactions were carried out as described above. Free and protein-bound DNAs were separated by electrophoresis on nondenaturing polyacrylamide gels and subjected to piperidine cleavage after elution. Cleaved products were separated on 12% polyacrylamide gels containing 7 M urea.

Cloning of a Full-length cDNA and Splice
Variants of NZF-1-Using radiolabeled ␤RARE DNA binding site as a probe, a 1.3-kb cDNA fragment was cloned from a rat pituitary cell line (GC) gt11 library by expression screening. Sequence analysis of this clone revealed the presence of an open reading frame containing two Cys-Cys, His-Cys type zinc fingers, which would be predicted to form a DNA binding motif. Because the 5Ј end of this cDNA did not contain an initiator methionine, five additional overlapping clones, two from rat pituitary cell line libraries, and three from a rat cerebellum library were obtained by DNA hybridization screening and sequenced (Fig. 1B), all proving to represent sequences from a single transcript. The assembled 4.7-kb NZF-1 cDNA sequence contained stop codons in all three reading frames preceding an ATG that initiated a 3.5-kb open reading frame, followed by a 360-bp 3Ј-untranslated region containing consensus polyadenylation signals.
The sequence predicted an 1187-amino acid protein that contained six Cys-Cys, His-Cys type zinc fingers, organized in clusters of two and three zinc fingers, separated by 329 amino acids, as well as a single zinc finger near the amino terminus (Fig. 1A). The amino acid sequences of the six zinc fingers share striking homology with each other (Fig. 1C). The NZF-1 protein contains a highly acidic region in the amino terminus (79% Asp or Glu, between amino acids 88 and 173), and a serine/threonine-rich sequence located between the two clusters of zinc fingers (35% Ser or Thr, between amino acids 625 and 715) (Fig. 1A).
We subsequently identified two alternatively spliced forms of NZF-1, which predict deletions of 63 and 135 nucleotides, removing coding information for the first and second zinc fingers, respectively. In an RNase protection assay, one of the splice forms is expressed at levels significant when compared to the predominant transcript encoding the larger 1187-amino acid protein (Figs. 1B and 2A and data not shown).
Neuronal-restricted Expression of NZF-1-Ribonuclease protection assays were employed to study the tissue distribution of NZF-1 messenger RNA. A wide variety of rat tissues were examined, including muscle, kidney, spleen, liver, heart, brain, skin, lung, eye, thymus, pancreas, adrenal, testis, and ovary. NZF-1 mRNA was detected only in brain, adult pituitary, and pituitary cell lines ( Fig. 2A). Because NZF-1 expression in testis was detected with a probe encompassing nucleotides 2979 -3366 (probe B), but not nucleotides 2058 -2664 (probe A), an alternatively spliced form of NZF-1 appears to be selectively expressed in the testis (Fig. 2B). This conclusion is consistent with RNA blot analysis, which revealed that transcripts detected in brain and testis using NZF-1 probe are of different size (data not shown). The levels of NZF-1 gene expression became progressively higher in adult as compared to neonatal pituitary (Fig. 2B), and the levels of NZF-1 expression were consistently higher in brain than those in pituitary and testis.
In order to determine the temporal and spatial expression patterns of NZF-1, we performed in situ hybridization experiments on both embryonic and adult rat tissues. As early as e11.5, NZF-1 expression was evident throughout the proliferating cortex neuroepithelium, the developing medulla, and in the spinal cord. At e12.5, NZF-1 mRNA was detected in the nasal epithelium as the nasal cavity forms (data not shown). At e13.5, NZF-1 expression was detected in the trigeminal ganglia, the dorsal root ganglia, and the ganglion cell layer of the retina (Fig. 3A). The expression of NZF-1 transcripts in the brain and the spinal cord reached the highest levels of detection at e14 -e15 and subsequently decreased slightly (Fig. 3 and data not shown). Later in brain development, transcripts of NZF-1 were detected in the cerebellar neuroepithelium, choroid plexus primordium, and within the cephalic flexure. NZF-1 transcripts were widely expressed in the adult brain, at levels lower than that detected during development. In situ hybridization experiments revealed that in adult testis, NZF-1 was expressed in the periphery of a subset of seminiferous tubules, indicating that the mRNA was present at early stages of the developing germ cell, i.e. in spermatogonia and/or early phase of spermatocytes I (data not shown). Thus, expression of the NZF-1 gene is restricted to the nervous system, pituitary gland, and testis, exhibiting highest levels of expression in the developing central nervous system.

NZF-1 Is a Member of a Gene
Family-Interestingly, an NZF-1 cDNA was also isolated by expression screening with a Pit-1 enhancer element (RDE) (17) from a pituitary cell line (GC) gt11 library, performed in an effort to search for transcription factors that interact with the Pit-1 gene enhancer. A second element in the Pit-1 gene enhancer, the pituitary specific element (PSE) (17), was used as a probe in expression screening of a mouse pituitary library, resulting in the identification of a cDNA encoding a highly related mouse zinc finger protein, referred to as NZF-2. Sequencing of fragments encompassing this clone revealed that it contains five zinc fingers corresponding to specific fingers in NZF-1 (Fig. 4A). This mouse clone has high homology to the human glia transcription factor referred to as MyT1, reported to bind to the proteolipid protein gene promoter (27).
Genomic Southern blot analysis was employed to determine whether NZF-1 and NZF-2 were encoded by distinct genes. Duplicate Southern blots hybridized with cDNA probes encoding homologous regions of NZF-1 and NZF-2 revealed distinct patterns for the two probes, indicating they are encoded by two different genes (Fig. 4B). The chromosomal localization of the two NZF genes were determined in mouse by linkage analysis of a backcross between strains C57BL/6J and M. spretus, with C57BL/6J as the recurrent parent. A screen of restriction enzymes revealed a restriction fragment length variant (RFLV) for the NZF-1 gene, designated Nzf-1, with the enzyme PvuII. Following Southern analysis, DNA from strain C57BL/6J yielded hybridized bands of 8.0, 5.2, and 2.7 kb, DNA from M. spretus yielded bands of 9.8 and 8.0 kb, and DNA from F1 hybrids contained all these bands (data not shown). The RFLV was scored in 49 backcross mice and compared to the segregation of about 350 previously typed genetic markers. Linkage was observed with several genetic markers on proximal mouse chromosome 12 (Fig. 5). An RFLV for the Nzf-2 gene was identified using the enzyme SstI. Thus, DNA from C57BL/6J mice exhibited hybridizing bands of 7.6 and 3.3 kb, DNA from M. spretus mice exhibited bands of 10.5, 3.3, and 2.3 kb, and F1 mice exhibited all four bands (data not shown). The RFLV exhibited close linkage with the marker D2Mit25 at the distal end of mouse chromosome 2 (Fig. 5).
DNA Binding Properties of the NZF-1 Protein-Based on the primary amino acid sequence of the NZF-1 protein, it was predicted that this protein would contain two clusters of zinc fingers capable of serving independently as DNA binding domains. We examined bacterially expressed glutathione S-transferase fusion proteins containing either the central region with one or two zinc fingers (1ZF and 2ZF), a carboxylterminal fragment spanning three zinc fingers (3ZF), or a region of NZF-1 spanning five zinc fingers (5ZF) (Fig. 6A). Although the peptide containing one zinc finger would still be expected to fold into the presumed ␤ sheet, ␣-helical DNA binding structure in the presence of zinc, this single zinc finger region (1ZF) exhibited only nonspecific DNA binding activity. However, all other protein segments encompassing either two, three, or five zinc finger motifs bound specifically to the ␤RARE DNA element (Fig. 6A).
In order to determine the specific nucleotide sequences that were critical for binding by these two or three zinc finger regions, we utilized synthetic double-stranded oligonucleotides encompassing wild-type and mutant ␤RARE using electrophoretic mobility shift assays. Systematic mutations altering this element revealed the critical nucleotides of GAAAGTT motif for binding by the 2ZF polypeptide. The region encompassing three zinc fingers bound an additional GTT motif 4 bp 5Ј of the GAAAGTT core (Fig. 6B), perhaps due to the ability of an additional finger to contact additional nucleotides, resulting in increased DNA specificity. We therefore conclude that for all elements examined, the core DNA element recognized by NZF-1 was GAAAGTT.
To further define this core binding element, a series of mutant oligonucleotides were tested for binding by the two-zinc finger segment. 32 P-Labeled ␤RARE oligonucleotide was mixed with 3-, 10-, or 50-fold molar excess of unlabeled competitor oligonucleotides before NZF-1 protein was added. These analysis identified that the AAGTT sequence was crucial for optimal binding and that purines at the two bases preceding this sequence are preferred for optimal binding by the NZF-1 zinc fingers (Fig. 7).
Electrophoretic mobility shift experiments confirmed that NZF-1 bound specifically to a series of DNA sites: Pit-1 PSE, Pit-1 RDE, and the pyridoxal phosphate promoter element (data not shown) in addition to the ␤RARE, and all four DNA elements used in expression cloning shared a highly homologous core motif (Fig. 7). NZF-1 and NZF-2 share a high degree of amino acid similarity in the zinc finger regions as well as in the carboxyl terminus and bind to the same sequences.
Analysis of methylation interference of binding by the methyl groups of the N-7 position of guanine and the N-3 position of adenines, indicative of major and minor groove contacts, respectively, was then utilized to investigate the specific contacts of the NZF-1 protein with target DNA elements. 32 P-Labeled probes (representing the ␤RARE and the Pit-1 PSE) were methylated with dimethyl sulfate and incubated with GST NZF-1. The pattern of inhibition of G residues in the major groove of the consensus core motifs and of A residues showing weaker signals in the bound complexes indicated that the core binding sequence was consistent with that defined by mutagenesis, electrophoretic mobility shift assays (Fig. 8).
Transactivation by NZF-1-In addition to DNA binding motifs, NZF-1 contained regions such as acidic and serine/threonine-rich sequences that have been commonly found in the activation domains of many transcription factors. CV-1 and HeLa cells, which do not contain detectable NZF-1 mRNA by RNase protection assay, were chosen to examine the potential transcriptional effects of NZF-1. Because NZF-1 was found to bind to both ␤RARE and Pit-1 enhancer elements in a sequence-specific manner, we tested whether NZF-1 was capable of transactivating promoters containing these elements. The complete NZF-1 cDNA was introduced into an expression vector under control of the Rous sarcoma virus promoter and co-transfected with the hRAR␤ promoter-luciferase reporter plasmid. NZF-1 moderately activated the hRAR␤ promoter, approximately 3-fold compared with the RSV vector control (Fig. 9A). When NZF-1 was co-transfected with a Pit-1 enhancer/promoter luciferase reporter gene in HeLa cells, NZF-1 activated the reporter 4-fold, and co-transfected NZF-1 synergized with Pit-1 (Fig. 9B). Thus, in this context, the NZF-1 protein is capable of serving as a co-regulator. Retinoic acid can regulate gene expression through the action of its receptors. However, NZF-1 neither synergized with retinoic acid receptor nor inhibited activation by retinoic acid of the hRAR␤ gene promoter (data not shown). It therefore appears that NZF-1 and retinoic acid can independently activate the hRAR␤ promoter.  A, electrophoretic mobility shift assays demonstrate that specific binding of NZF-1 to DNA requires a minimum of two zinc fingers. NZF-1 was expressed as GST fusion proteins containing either one (1ZF), two (2ZF), three (3ZF), or five (5ZF) zinc fingers and incubated with a 32 P-labeled ␤RARE probe. In the presence of competitor DNA, only 2ZF, 3ZF, and 5ZF bind ␤RARE specifically. B, 2ZF and 3ZF recognize similar DNA sequences while 3ZF has more specificity. Only the bound complexes are shown. DISCUSSION We have identified a 4730-base pair cDNA encoding a novel 1187-amino acid protein, NZF-1, containing six Cys-Cys, His-Cys type zinc fingers, organized as internal clusters of two and three fingers, with one finger located in the amino terminus. The expression of this factor is highly restricted to the nervous system, pituitary gland, and testis with highest levels of expression in the developing nervous system.
The primary amino acid sequences of all component zinc fingers are very homologous. Because of the spacing between the Cys and His residues, the folding of the NZF-1 zinc finger motifs is likely to more closely resemble that of the Cys-Cys, His-His type of zinc fingers than the Cys-Cys, His-Cys fingers of retrovirus proteins, such as the nucleic acid-binding protein of Rauscher murine leukemia virus (28).
Each zinc finger cluster forms an independent DNA binding domain and recognizes similar specific target sequences, although the three zinc finger domain demonstrates higher binding specificity. Crystallographic studies on the Cys-Cys, His-His class of zinc fingers has revealed that two zinc fingers make major groove contacts. The third and fourth zinc fingers in the same cluster make minor groove contacts (29). A similar mechanism may operate to generate more selective binding site usage for the three zinc finger region.
Several transcription factors that contain two independent regions of zinc finger DNA binding domains have been described (28,30). In each case, the amino acid sequences of the two separate DNA binding domains are very homologous to each other, and the target sequences they recognize are also similar. Although the full biological significance of such dual DNA binding domains is not known, it raises the possibility that a single protein could simultaneously bind to two significantly spaced DNA sequences and approximate the DNA sequences.
Although NZF-1 is potentially expressed at low levels in embryonic pituitary, significant levels of NZF-1 transcripts are present in the adult anterior pituitary gland. In vitro, NZF-1 specifically binds the Pit-1 enhancer and transactivates the Pit-1 enhancer/promoter reporter genes, making it a candidate for modulation of Pit-1 gene regulation in vivo.
In the course of these studies, we isolated a related mouse gene, named NZF-2, which exhibits high homology to the human glia transcription factor MyT1 (27). Because of the Cys-Cys, His-Cys configuration, these two proteins form a subclass of the most common zinc finger transcription factors, and the extensive homology in both zinc finger regions and in the carboxyl terminus suggests that these factors might exert similar and overlapping functions, acting in distinct cell types in the central nervous system.
The genes for NZF-1 and NZF-2, designated Nzf-1 and Nzf-2, respectively, were mapped in the mouse by linkage analysis of an interspecific cross. Nzf-1 is located on mouse chromosome 12 in a region that appears to be syntenic with human chromosome 2p. Nzf-2 is located on distal mouse chromosome 2 in a region syntenic with human chromosome 20q. Neither region in the mouse contains mapped mutations that are likely to be relevant to these genes.
Thus, a new family of Cys-Cys, His-Cys proteins, expressed FIG. 8. Methylation interference analysis. Oligonucleotides were labeled at the 5Ј end using T4 kinase and [ 32 P]ATP. After annealing, double-stranded probes were gel-purified and methylated with dimethyl sulfate. Binding reactions were carried out as described above. Free and protein-bound DNAs were separated by electrophoresis on nondenaturing polyacrylamide gels and subjected to piperidine cleavage after elution. Cleaved products were separated on 12% polyacrylamide gels containing 7 M urea. Methylated guanine and adenine residues that interfere with binding by NZF-1 are indicated by arrowheads. A, the sense and antisense strands of ␤RARE. B, the sense strand of the Pit-1 PSE enhancer element.
FIG. 9. Transcriptional activity of NZF-1. A, human ␤RAR promoter/luciferase reporter gene was co-transfected into CV-1 cells with RSV vector or RSV NZF-1 expression plasmids. Luciferase activity of the control RSV vector co-transfection was set at 1. B, mouse Pit-1 enhancer/promoter/luciferase reporter was co-transfected into HeLa cells with RSV vector, RSV NZF-1, or RSV NZF-1 and RSV Pit-1. Luciferase activity for RSV vector co-transfection was set at 1.
in the nervous and endocrine systems, has been identified, and these factors are likely to exert selective functions in development and homeostasis of the neuroendocrine system.