ABSTRACT
Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPalpha, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPalpha in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPalpha is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPalpha has no affinity for these E2F sites, but when recombinant C/EBPalpha is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPalpha protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPalpha directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPalpha can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPalpha-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins/genetics , Gene Expression Regulation , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Animals , CCAAT-Enhancer-Binding Proteins , Cell Division/genetics , Cell Line , E2F Transcription Factors , E2F1 Transcription Factor , Mice , Protein Isoforms/genetics , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1ABSTRACT
We recently cloned a cDNA for CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis or Batten disease. To resolve the genomic organization we used a cosmid clone containing CLN3 to sequence the entire gene in addition to 1.1 kb 5' of the start of the published CLN3 cDNA and 0.3 kb 3' to the polyadenylation site. CLN3 is organized into at least 15 exons spanning 15 kb and ranging from 47 to 356 bp. The 14 introns vary from 80 to 4227 bp, and all exon/intron junction sequences conform to the GT/AG rule. Numerous repetitive Alu elements are present within the introns and 5'- and 3'-untranslated regions. The 5' region of the CLN3 gene contains several potential transcription regulatory elements but no consensus TATA-1 box was identified. CLN3 is homologous to 27 deposited human ESTs, and sequence comparisons suggest alternative splicing of the gene and the existence of transcribed sequences upstream to the start of the published CLN3 cDNA.
Subject(s)
Genes/genetics , Membrane Glycoproteins , Molecular Chaperones , Neuronal Ceroid-Lipofuscinoses/genetics , Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Exons/genetics , Gene Expression , Humans , Introns/genetics , Molecular Sequence Data , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
A yeast artificial chromosome (YAC) contig has been constructed in 16p12.1-p11.2 that encompasses three loci (D16S288, D16S299, and D16S298) closely linked to the gene causing Batten disease or juvenile-onset neuronal ceroid lipofuscinosis (CLN3). The physical map has been ordered using 42 sequence tagged sites. Four genes, interleukin-4 receptor (IL4R), phenol-preferring phenol sulfotransferase (STP), monoamine-preferring phenol sulfotransferase (STM), and sialophorin (SPN), have been mapped to the YAC contig. A partial genomic restriction map has been constructed to confirm the order and distances between D16S298, predicted to be the locus closest to CLN3. The overlapping genomic clones are a valuable resource for cloning the Batten gene (CLN3) and other genes in the region.
Subject(s)
Chromosomes, Human, Pair 16 , Neuronal Ceroid-Lipofuscinoses/genetics , Antigens, CD/genetics , Arylsulfotransferase/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Artificial, Yeast , Cosmids , DNA Primers , Genetic Linkage , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Interleukin-4/metabolism , Isoenzymes/genetics , Leukosialin , Molecular Sequence Data , Polymerase Chain Reaction/methods , Receptors, Interleukin/genetics , Receptors, Interleukin-4 , Restriction Mapping , Sequence Tagged Sites , Sialoglycoproteins/geneticsABSTRACT
In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes.
