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M Yaniv

Publications and source records attributed to M Yaniv.

At least 145 records · Page 8Linked to original sources
PubMed1988

Several distinct "CCAAT" box binding proteins coexist in eukaryotic cells.

Even though a "CCAAT" box element is localized approximately equal to 80 base pairs upstream of the transcription initiation site in many eukaryotic promoters, it is not recognized by a single ubiquitous transcription factor. We show here that such a sequence present in the rat albumin promoter binds a protein distinct from the CCAAT-binding transcription factor CTF/NFI or from the CCAAT-binding protein (CBP) previously described. The protein binding to the albumin CCAAT sequence (ACF or albumin CCAAT factor) is not exclusive to liver, since we found a protein with identical properties in spleen.

Albumins↗
PubMed1988

Use of retroviral vectors for mapping of splice sites in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5' end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

Base Sequence↗
PubMed1988

A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells.

We have identified and characterized two mutually exclusive nuclear proteins that interact with a single crucial element of the albumin promoter. One, albumin proximal factor (APF), is found only in liver or differentiated hepatoma cells and is probably identical to the liver-specific factor named HNF1, alpha 1TFB, or HP1-binding protein. The other, variant albumin proximal factor (vAPF), is present in dedifferentiated hepatoma cells as well as in somatic cell hybrids that show extinction of the expression of liver-specific proteins, including albumin. Reversion to the hepatic phenotype of either a dedifferentiated variant or an extinguished somatic hybrid clone is accompanied by loss of vAPF and reappearance of APF. These two proteins differ in their thermostability and in their molecular weight, while displaying identical sequence specificities. Both proteins interact with a homologous motif present in promoter regions of several other liver-specific genes. In vitro transcription assays, using a rat liver nuclear extract, indicate that the binding of APF to its target sequence is required for albumin transcription. These results suggest that a modification in the primary structure of a transcription factor is correlated with the differentiated state of the hepatic cell.

Albumins↗
PubMed1988

Interplay of viral and cellular proteins along the long control region of human papillomavirus type 18.

The long control region of human genital papillomavirus type 18 harbors transcription regulatory elements, such as the E6 promoter and a cell type-specific enhancer independent of the E2 protein. By performing DNase I footprint experiments in vitro with protein extracts from different cell lines and tissues we searched for cellular factors interacting with the totality of the control region. We detected a total of eight different protected sites; most of them were found with all the extracts. Two of these sites, one in the enhancer and the other in the sequences proximal to the E6 cap site, interact with a member of the activator protein 1 family. However, in the absence of fine mutational analysis, we cannot readily discern an exact functional role for the different binding sites. We also characterized two regions, which are protected only in the presence of E2, corresponding to the perfect palindromes ACCGN4CGGT and present either 500 nucleotides or tandemly repeated about 70 nucleotides upstream of the E6 cap site. This last protected area covers a large part of the putative TATA box of the E6 promoter and could explain its repression by bovine papilloma virus type 1 E2, which could interfere with binding of the TFII D ubiquitous transcription factor to the TATA sequence of the promoter.

Base Sequence↗
PubMed1988

Study of the E2 gene product of the cottontail rabbit papillomavirus reveals a common mechanism of transactivation among papillomaviruses.

The long control region (LCR) of the cottontail rabbit papillomavirus (CRPV) harbors a transcriptional promoter which can be transactivated, as reflected by cat gene expression, by cotransfection with plasmids which express the intact E2 open reading frame of CRPV, human papillomavirus type 18 (HPV18), and bovine papillomavirus type 1 (BPV1). The E2 protein of CRPV can also transactivate the LCRs of BPV1, HPV1, and HPV18 inserted in front of the cat gene in enhancer or promoter configuration. Competition experiments in vivo and binding studies with CRPV E2 protein synthesized in vitro suggest that the different E2 proteins transactivate transcription by a common mechanism involving binding to the same ACCG-CGGT target sequence. The C-terminal part of the protein is necessary for its DNA-binding function. Analysis of the transactivation data and of the LCR sequences of these four viruses suggests that the two cutaneous viruses (CRPV and BPV1) present a similar pattern of promoter regulation but that the activity of the promoters of genital human viruses is less dependent on E2 regulation and is at least partially regulated by cellular factors.

Animals↗
PubMed1987

Factors involved in control of tissue-specific expression of albumin gene.

Elements controlling tissue-specific expression of the rat albumin gene reside within roughly 150 bp upstream of the transcriptional initiation site. We show here by DNAase I footprinting assays that at least four distinct factors present in extracts derived from cells expressing albumin interact with these sequences. One of these factors is closely related or identical to nuclear factor 1 (NF1). Extracts from nonhepatic tissues or from dedifferentiated hepatoma cells that do not transcribe the albumin gene display a different pattern of DNA-protein interactions. Mixing experiments show that the variant pattern of dedifferentiated cells is dominant over that of differentiated ones, suggesting that nonexpressing cells contain dominant negatively acting factors.

Albumins↗
PubMed1987

Enhancer-mediated activation of a growth-regulated promoter.

We have demonstrated that the collagen alpha 2 type 1 promoter inserted in an expression vector, behaves as a growth-regulated promoter, which is consistent with previous observations that collagen synthesis is growth-regulated in vivo. In contrast, the activity of the H2-K or the simian virus 40 early promoters does not seem to be affected by the rate of cell proliferation. The insertion of a polyoma enhancer 5' or 3' to the collagen transcription unit activates the collagen alpha 2 type 1 promoter, by a threefold greater factor in slowly growing cells compared to cells growing exponentially. These results show that enhancers can also function in slowly proliferating cells and activate the normally low activity of a promoter in these cells.

Animals↗
PubMed1987

Two different factors bind to the alpha-domain of the polyoma virus enhancer, one of which also interacts with the SV40 and c-fos enhancers.

Two nuclear factors from mouse 3T6 cells bind to a 22-bp segment constituting the alpha-domain of the polyoma virus enhancer. Binding of each factor can be competed out selectively by the appropriate double-stranded oligonucleotide, indicating that this binding is not strictly cooperative. Sequence homology between the two binding sites and the similar size of the protected regions may indicate that both factors, PEA1 and PEA2, are closely related. The binding site of PEA1 is centered on a sequence showing strong homology to the SV40 enhancer, the binding site of PEA2 is located immediately adjacent to it and shows a strong homology to the c-fos enhancer. Surprisingly, both SV40 and c-fos enhancers interact with PEA1, probably due to the presence of an extra base pair relative to c-fos in the PEA2 site. Factor PEA1 is probably identical to the recently described activator protein 1 (AP1).

Animals↗
PubMed1987

The BPV1-E2 trans-acting protein can be either an activator or a repressor of the HPV18 regulatory region.

The human papillomavirus 18 (HPV 18) long control region contains promoter and enhancer elements whose activity is restricted to several human cell lines of epithelial origin. This enhancer possesses a considerable constitutive activity which is further stimulated in the presence of the E2 trans-activating protein of bovine papillomavirus 1 (BPV1). Surprisingly the same BPV1 protein strongly repressed transcription from the genuine HPV18 enhancer-promoter DNA sequences. We suggest that binding of several molecules of E2 protein between the viral CAAT and TATA elements sterically hinders transcription initiation from this promoter, while the same DNA--protein assembly stimulates the SV40 promoter when cloned in an enhancer configuration upstream of this heterologous promoter. Unlike BPV1-E2 the homologous E2 gene product does not seem to strongly modulate viral transcription. Finally the BPV1-E2 gene product may repress some essential viral or host genes, since we failed to isolate HeLa cells expressing BPV1-E2.

Cell Line↗
PubMed1987

gal4 transcription activator protein of yeast can function as a repressor in Escherichia coli.

The chromosomal lac operator of Escherichia coli was replaced by a 22 bp oligonucleotide containing the binding site of the yeast gal4 protein. Induction of gal4 protein synthesis in these bacteria repressed beta-galactosidase synthesis at least 30-fold. These results show that it is possible to detect in bacteria with a simple assay the DNA binding activity of a eukaryotic protein with a defined sequence specificity. This opens new avenues for the isolation in E. coli of mutants of DNA binding proteins unable to bind to their DNA targets, and for direct cloning in bacteria of cDNA coding for DNA binding proteins with defined sequence specificity.

Escherichia coli↗
PubMed1987

Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens.

RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.

Acetyltransferases↗
PubMed1987

Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.

Acetyltransferases↗
PubMed1986

Molecular analysis of the interaction between an enhancer binding factor and its DNA target.

The fine contacts of a mouse nuclear factor, called PEB1, with the B enhancer of polyoma virus were analyzed. It protects against DNaseI attack a region of about 50 base pairs that can be divided in two domains. The first contains a GC-rich palindrome and the homology to the SV40 enhancer. The second is homologous to a sequence in the immunoglobulin (Ig) heavy chain gene enhancer. Methylation interference and protection experiments reveal strong specific contacts only with a purine rich track on the late coding strand of the early proximal part of the palindrome. Deletion analysis show that the minimal sequences necessary for binding include only the first domain. The Ig homology contributes only weakly to the binding. The minimal core is similar to the core of the B enhancer defined in vivo. The interactions we observe here are reminiscent of those of TFIIIA positive transcription factor and the 5SRNA gene of Xenopus.

Animals↗
PubMed1986

Production of spliced DNA copies of the cottontail rabbit papillomavirus genome in a retroviral vector.

The early region of the cottontail rabbit papillomavirus (CRPV) genome has been introduced into a retroviral vector and recombinant retroviruses, produced upon transfection of the psi 2 packaging cell line, have been used to infect NIH 3T3 cells. Spliced derivatives of the CRPV early region can be rescued from the infected cells. Sequence analysis demonstrates that the major splicing event observed in RNA in tumours is faithfully reproduced in this system. This splice generates a polycistronic mRNA that contains in its 5' portion the E7 open reading frame, or both E6 and E7, and at its 3' end a reading frame with codons for three amino acids from the N-terminus of E1 linked to codons for 100 amino acids from the C-terminus of the E4 region. Recombinant retroviruses containing intact or spliced CRPV sequences can now be used to introduce the viral genes efficiently into a variety of cell lines.

Animals↗
PubMed1986

Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice.

Transgenic mice carrying the H-2K/human growth hormone (hGH) fusion gene were produced by microinjecting into the pronucleus of fertilized eggs DNA molecules containing 2 kb of the 5' flanking sequences (including promoter) of the class I H-2Kb gene joined to the coding sequences of the hGH gene. Thirteen transgenic mice were obtained which all contained detectable levels of hGH hormone in their blood. Nine grew larger than their control litter-mates. Endogenous H-2Kb and exogenous hGH mRNA levels were analysed by S1 nuclease digestion experiments. hGH transcripts were found in all the tissues examined and the pattern of expression paralleled that of endogenous H-2K gene expression, being high in liver and lymphoid organs and low in muscle and brain. Thus 2 kb of the 5' promoter/regulatory region of the H-2K gene are sufficient to ensure regulated expression of hGH in transgenic mice. This promoter may therefore be of use to target the expression of different exogenous genes in most tissues of transgenic mice and to study the biological role of the corresponding proteins in different cellular environments.

Animals↗
PubMed1986

Structure of transcriptionally active chromatin.

Transcriptionally active or potentially active genes can be distinguished by several criteria from inactive sequences. Active genes show both an increased general sensitivity to endonucleases like DNase I or micrococcal nuclease and the presence of nuclease hypersensitive sites. Frequently, the nuclease hypersensitive sites are present just upstream of the transcription initiation site covering sequences that are crucial for the promoter function. Viral or cellular transcription enhancer elements are also associated with DNase I hypersensitive sites. At least for the SV40 enhancer, it was shown by electronmicroscopic studies that the DNase I hypersensitive DNA segment is excluded from nucleosomes. It is highly plausible that the binding of regulatory proteins to enhancer or promoter sequences is responsible for the exclusion of these DNA segments from nucleosomes and for the formation of nuclease hypersensitive sites. We speculate that the binding of such proteins may switch on a change in the conformation and/or the protein composition of a chromatin segment or domain containing one to several genes. Biochemical analysis of fractionated nucleosome particles or of active and inactive chromatin fractions have revealed differences in the composition as well as in the degree of modification of histones in these two subfractions of the chromosome. However, until present it is impossible to define unambiguously what are the crucial structural elements that distinguish between particles present on active and inactive chromatin.

Animals↗
PubMed1985

Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine-free medium.

The lysA gene of Escherichia coli encodes for a diaminopimelic acid decarboxylase (EC 4.1.1.20) which allows the conversion of diaminopimelic acid into lysine in bacteria. It has been cloned in an eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse alpha-globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) has been introduced into several mammalian cell lines by cotransfection with a second selectable marker i.e. the polyoma-transforming DNA. Selection for morphologically transformed rat cells which contained the intact lysA sequences, allowed the determination of the concentration of diaminopimelic acid in the lysine-free medium that permitted cell growth. lysA-expressing clones were directly selected in a medium containing 10 mM diaminopimelic acid, after transfection with pSB99 alone. Southern blot analysis on selected clones have shown that they contain up to 30-50 integrated copies of the plasmid in tandem arrangement. Finally, we demonstrated that lysA-expressing clones incorporate a significant amount of radiolabelled [3H]diaminopimelic acid in acid-insoluble material. The recombinant plasmid can serve as a selectable marker, in growth medium in which lysine was replaced by its direct bacterial precursor.

Animals↗