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C M Sax

Publications and source records attributed to C M Sax.

32 records · Page 2Linked to original sources
PubMed1993

Binding of tissue-specific forms of alpha A-CRYBP1 to their regulatory sequence in the mouse alpha A-crystallin-encoding gene: double-label immunoblotting of UV-crosslinked complexes.

The alpha A-CRYBP1 regulatory sequence (alpha A-CRYBP1RS), at nucleotides -66 to -57 of the mouse alpha A-crystallin-encoding gene (alpha A-CRY) promoter, is an important control element involved in the regulation of mouse alpha A-CRY expression. The gene encoding a protein (alpha A-CRYBP1) that specifically binds to the alpha A-CRYBP1RS sequence has been cloned from a cultured mouse lens cell line. In the present study, we have used an antibody (specific to the alpha A-CRYBP1 protein and made against a synthetic peptide) to directly identify UV-crosslinked protein-DNA complexes via a double-label immunoblotting technique. Multiple alpha A-CRYB1 antigenically related proteins interacted with alpha A-CRYBP1RS in nuclear extracts from both a cloned mouse lens cell line (alpha TN4-1) that expresses alpha A-CRY and a mouse fibroblast line (L929) that does not express the gene. Two sizes (50 kDa and 90 kDa) of proteins reacting with the alpha A-CRYBP1-specific Ab were detected in both cell lines and, in addition, a > 200-kDa protein reacting with the Ab was unique to the fibroblast line. Thus, alpha A-CRYBP1 antigenically related proteins interact with alpha A-CRYBP1RS regardless of alpha A-CRY expression. Moreover, differential processing of the alpha A-CRYBP1 protein and/or alternative splicing of the alpha A-CRY transcript may affect expression of alpha A-CRY.

Alternative Splicing↗
PubMed1993

Functional analysis of chicken vimentin distal promoter regions in cultured lens cells.

Synthesis of the cytoskeletal intermediate filament protein vimentin (Vim) in the lens is unexpected due to the mesenchymal preference of Vim-encoding gene (Vim) expression and the epithelial origin of the lens. Previous studies indicated that chicken Vim gene expression in cultured lens cells is regulated by both positive- and negative-acting sequence elements within the first -767 nucleotides (nt) of its promoter. Here, we demonstrate the existence of additional upstream chicken Vim promoter elements which function in transfected lens cells. Sequences within the nt -1360/-1156 region repressed promoter activity in transfected lens cells to levels lower than that observed for the previously defined more proximal repressor elements. The -1612/-1360 region activated promoter activity to levels similar to those observed for the strongest previously defined proximal promoter. The nt sequence analysis of the upstream promoter region revealed the presence of multiple consensus repressor and activator transcription-factor-binding sites. Several of these sites have been implicated for lens expression of enzyme-crystallin-encoding genes (cry), suggesting that Vim expression may share features with the cry genes for recruitment and high-level expression in the lens.

Animals↗
PubMed1993

Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.

Animals↗
PubMed1993

Protein-DNA interactions of the mouse alpha A-crystallin control regions. Differences between expressing and non-expressing cells.

Genomic footprinting, in vitro footprinting and mobility shift assays were used to investigate the molecular basis for expression of mouse alpha A-crystallin, a major structural protein of the transparent lens of vertebrates. The putative control region of the mouse alpha A-crystallin gene was footprinted by DNase I digestion in nuclear extracts, by dimethylsulfate treatment in cultured cells, and by micrococcal nuclease digestion in isolated nuclei. The resulting digestion patterns were compared between alpha TN4-1 lens cells, which express alpha A-crystallin, and L929 fibroblasts, which do not express alpha A-crystallin. Four regions of DNA were found occupied in both cell types. These included positions -111 to -97 (DE-1 region), positions -75 to -55 (alpha A-CRYBP1 region), positions -35 to -12 (TATA box and PE-1 region), and positions +23 to +43 (an AP-1 consensus sequence). The DNase I footprints of the DE-1 and alpha A-CRYBP1 regions, previously implicated as functional control elements, were substantially more pronounced using nuclear extract from the alpha TN4-1 cells than from the L929 fibroblasts, suggesting more stable protein binding with the former than with the latter. Numerous in vivo binding variations were noted between the two cell types in all four of the footprinted regions examined. Finally, two complexes (A and B) were formed specifically with nuclear extracts from the alpha TN4-1 cells and a synthetic deoxyoligonucleotide comprising the alpha A-CRYBP1 region. These data indicate that specific differences in protein-DNA interactions with putative control regions are associated with tissue-preferred expression of the mouse alpha A-crystallin gene.

Actins↗
PubMed1992

Conservation of mouse alpha A-crystallin promoter activity in chicken lens epithelial cells.

Previous transfection experiments have shown that 162 base pairs (bp) of the 5' flanking sequence of the chicken alpha A-crystallin gene are required for promoter activity in primary chicken lens epithelial cells (PLE), while only 111 bp of the 5' flanking sequence are needed for activity of the mouse alpha A-crystallin promoter in transfected chicken PLE cells or in a SV40 T-antigen-transformed transfected mouse lens epithelial cell line (alpha TN4-1). The effect of site-directed mutations covering positions -111 to -34 of the mouse alpha A-crystallin promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was compared in transfected chicken PLE cells and mouse alpha TN4-1 cells; selected mutations were also examined in a nontransformed rabbit lens epithelial cell line (N/N1003A). In general, the same mutations reduced promoter activity in the transfected lens cells from all three species, although differences were noted. The mutations severely affected regions -111/-106 and -69/-40 regions in all the transfected cells examined; by contrast, mutations at positions -105/-99 and -87/-70 had a somewhat greater effect in the chicken PLE than the mouse alpha TN4-1 cells, while mutations of the -93/-88 sequence reduced expression in the alpha TN4-1 but not the PLE cells. A partial cDNA with sequence similarity to alpha A-CRYPB1 of the mouse has been isolated from a chicken lens library; mouse alpha A-CRYBP1 is a putative transcription factor which binds to the -66/-55 sequence of the mouse alpha A-crystallin promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Regulation of vimentin gene expression in the ocular lens.

Vimentin expression in the lens is striking due to the reported mesenchymal preference of vimentin and the epithelial origin of the lens. The amount of chicken vimentin mRNA levels determined by Northern blot analysis increased 3-fold from 7 to 14 days of embryonic lens development and then decreased 10-fold at 16 days of development, suggesting that post-transcriptional processes may contribute to the level of cytoplasmic vimentin mRNA during lens development. To analyze the mechanisms governing vimentin gene expression in the lens at the level of transcription, a series of chicken vimentin 5'-flanking region deletions were fused to the bacterial CAT gene and transfected into fibroblasts and lens cultures derived from three species. The -160 to +1 sequence conferred equal promoter activity in cultured chicken lens epithelial cells and fibroblasts. The -321 to -160 sequences increased promoter activity in all cultures, but more strongly in fibroblasts than in lens cells. Sequence elements in the region -608 to -321 repressed promoter activity in lens cells and fibroblasts. Promoter activity was partially restored in fibroblasts but not in lens cells by -767 to -608 sequences. Vimentin gene expression in the lens thus appears to be controlled by multiple positive- and negative-acting elements in its 5'-flanking sequence.

Animals↗
PubMed1990

Species-specific lens activation of the thymidine kinase promoter by a single copy of the mouse alpha A-CRYBP1 site and loss of tissue specificity by multimerization.

One copy of the mouse alpha A-crystallin gene alpha A-CRYBP1 site activated the thymidine kinase (tk) promoter in a mouse lens epithelial cell line but not in primary chicken lens cells; multiple copies further activated the tk promoter and extended expression to fibroblasts, B cells, and chicken lens cultures. The loss of lens specificity by multimerization may place selective constraints on the number of alpha A-CRYBP1 sites in the alpha A-crystallin promoter.

Animals↗
PubMed1990

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

Animals↗
PubMed1990

Regulation of the mouse alpha A-crystallin gene: isolation of a cDNA encoding a protein that binds to a cis sequence motif shared with the major histocompatibility complex class I gene and other genes.

We have shown by site-directed mutagenesis that the sequence between positions -69 and -40 of the mouse alpha A-crystallin gene is crucial for tissue-specific gene expression in a transfected mouse lens epithelial cell line transformed with the early region of simian virus 40. Gel retardation experiments with synthetic oligodeoxynucleotides revealed a mouse lens nuclear protein which bound specifically to the palindromic sequence 5'-GGGAAATCCC-3' at positions -66 to -57 in the alpha A-crystallin promoter. By screening a bacteriophage lambda gt11 expression library of the transformed lens cells, we isolated a 2.5-kilobase-pair cDNA encoding a fusion protein which bound to this sequence and to the regulatory element of the major histocompatibility complex (MHC) class I gene. This cDNA hybridized to a 10-kilobase-pair polyadenylated RNA present in many different tissues, including lens. It encoded a protein, tentatively called alpha A-CRYBP1, containing at least two zinc fingers. alpha A-CRYBP1 is either homologous or very similar to the human nuclear proteins MBP-1 (Baldwin et al., Mol. Cell. Biol. 10:1406-1414, 1990), PRDII-BFI (Fan and Maniatis, Genes Dev. 4:29-42, 1990), and HIV-EP1 (Maekawa et al., J. Biol. Chem. 264:14591-14593, 1989), which bind to regulatory elements of the MHC class I, beta interferon, and human immunodeficiency virus genes, respectively. Our results suggest that the lens-specific alpha A-crystallin, MHC class I, beta interferon and other genes have a similar cis-acting DNA regulatory motif that shares alpha A-CRYBPI, MBP-1, PRDII-BF1, HIV-EP1, or other closely related proteins as trans-acting factors.

Amino Acid Sequence↗
PubMed1989

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5'-flanking sequence.

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5'-end Vim::cat constructs demonstrate that this repression is controlled by a 5'-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to +71) which do not contribute to vimentin's down-regulation during myogenesis.

Animals↗
PubMed1988

Multiple elements are required for expression of an intermediate filament gene.

The expression of vimentin is unique within the intermediate filament multigene family. It is the only member which deviates from its usual tissue-specific expression pattern and whose 5'-flanking region contains multiple GC boxes, the binding site for Sp1. The activity of vimentin 5'-end:CAT fusions has been compared in cells where vimentin is highly expressed (mouse L cells) or not expressed at all (MH1C1). In addition, CAT activity has been examined by microinjection into Xenopus oocytes. Both in vivo expression and in vitro binding studies implicate Sp1 as a general regulatory factor in vimentin gene expression. Increased expression of 5'-end:CAT fusions in mouse L cells suggests that a fibroblast-specific enhancer element resides in the region -321 to -160. Low transcriptional activity in MH1C1 cells may be due to either the lack of this positive transcription factor(s) or the presence of a repressor element. Here, we demonstrate that the unique and complex pattern of vimentin gene expression is controlled by multiple cis-acting elements.

Animals↗
PubMed1987

The chicken vimentin gene. Nucleotide sequence, regulatory elements, and comparison to the hamster gene.

Here we report the nucleotide sequence of the chicken vimentin gene and its deduced primary amino acid sequence. A comparison of this gene to other intermediate filament protein genes demonstrates that both exon size and position are strongly conserved features of this multigene family. In addition, the hamster and chicken vimentin genes exhibit strong identity at the level of nucleotide (74%) and amino acid (80%) sequence. Interestingly, 40% of total sequence diversity is localized to the N terminus or "head" region of these genes whereas other protein domains (rod and C terminus) are remarkably identical in both nucleotide (81%) and amino acid (89%) sequence. Even stronger amino acid identity (100%) is exhibited in certain subdomains which may define regions crucial for filament formation and function. Not surprisingly, vimentin is more homologous across animal species than it is to other intermediate filament protein members (e.g. desmin) within the same species. A comparison of 5'-flanking sequences of the hamster and chicken genes as well as other characterized promoter elements (SV40, HSV-TK) reveals homologous sequence elements which may define common and/or unique sites involved in the modulation of gene expression. The implications of these sequence elements for both tissue-specific and developmental expression of the vimentin gene are discussed.

Amino Acid Sequence↗
PubMed1986

Craniofrontonasal dysplasia: clinical and genetic analysis.

We have identified a case of craniofrontonasal dysplasia which demonstrates the potential lethality of this gene. Genetic analysis of this pedigree and nine others reveals that craniofrontonasal dysplasia does not follow a Mendelian mode of inheritance and may be a human mutation analogous to the T-locus of mice.

Abnormalities, Multiple↗