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M M Zakin

Publications and source records attributed to M M Zakin.

At least 55 records · Page 3Linked to original sources
PubMed1993

Liver-enriched HNF-3 alpha and ubiquitous factors interact with the human transferrin gene enhancer.

The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated Ia. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver-enriched transcription factor HNF-3 alpha interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF-3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.

Base Sequence↗
PubMed1993

Transferrin gene as a model for liver-specific gene expression.

The results obtained in the recent years have led to an understanding of the mechanisms controlling liver-specific gene expression. They indicate that this expression needs an appropriate combination of hepatocyte and ubiquitous transcriptional factors, interacting with cis-acting DNA elements, in a characteristic manner for each gene. However, a number of important questions about liver organogenesis and specific cell differentiation remain to be answered.

Animals↗
PubMed1992

Identification of an enhancer involved in the melanoma-specific expression of the tumor antigen melanotransferrin gene.

Melanotransferrin (MTf) is a tumor associated antigen found in abundance on the surface of melanoma cells. It is a transferrin-like protein found in low amount in most adult tissues and whose gene is reminiscent of house-keeping genes. With the goal of understanding the regulatory mechanisms which might explain the enhancement of expression in tumor cells, we report here the characterization of a regulatory element located 2 kbp upstream from the promoter and whose deletion specifically impairs gene expression in melanoma cells; we show that this element is part of an enhancer composed of two modules which are each the target for the AP1 transcription factor. The two modules present a synergistic mode of action specific for melanoma cells which requires both of the 130 bp away AP1 sites. Furthermore, we show that the enhancer behaves differently according to the promoter context.

Antigens, Neoplasm↗
PubMed1992

Regulation of transferrin gene expression.

Transferrin (Tf) is the iron-transport protein of vertebrate serum. It is essentially synthesized in the liver, but lower amounts are also produced in other organs, such as testis and brain. A number of studies have been done to characterize the transcriptional elements implicated in the regulation of Tf gene expression in different organs. The results of these studies support the hypothesis that the Tf gene makes use of different combinations of nuclear proteins in different subsets of cells to achieve tissue-specific expression. It is interesting to point out that this occurs in tissues arising from different embryological origin.

Animals↗
PubMed1991

Characterization of the active part of the human transferrin gene enhancer and purification of two liver nuclear factors interacting with the TGTTTGC motif present in this region.

The human transferrin gene enhancer is composed of two functional domains (A and B). We have previously shown that domain A is able to mediate enhancer activity in transient expression experiments. Here, we show that multimers of the domain A single enhanson coupled to a canonical TATA box are sufficient to promote transcription in vitro with liver nuclear extracts. Gel mobility shift assays reveal the binding of two liver nuclear factors to this enhanson, and methylation interference experiments show that the motif 5'-TGTTTGCTTT-3' is the target site for these factors. This was confirmed by the use of mutants in gel retardation assays and transient expression experiments. The two proteins interacting with the decanucleotide have been purified from rat liver nuclear extracts by DNA affinity chromatography. The purified proteins named enhancer-binding protein (EBP)-45 and EBP-40 appear as single polypeptide bands with respective molecular masses of 45 and 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The TGTTTGC motif was found to be important for the hepatocyte-specific expression of several genes; and in some cases, it was demonstrated that the transcription factor CCAAT/enhancer-binding protein (C/EBP) is able to bind to this sequence. In vitro experiments show that EBP-45 and EBP-40 are different from C/EBP; they also show that the two proteins interact with the TGTTTGC motif present in control regions of other hepatic genes, such as the mouse albumin enhancer eH and hepatitis B virus enhancer E elements.

Animals↗
PubMed1991

Cloning and sequencing of PYBP, a pyrimidine-rich specific single strand DNA-binding protein.

In the human transferrin gene promoter, PRI and DRI are positive cis-acting elements interacting respectively with two families of proteins, Tf-LF1 and Tf-LF2. In this paper, we report the purification from rat liver nuclei, of one of these factors, PYBP, as well as the cloning and the sequencing of its cDNA. PYBP is a DNA-binding protein, purified as a 58 kDa doublet which binds only to single strand pyrimidine-rich DNA present for example in PRI and DRI. The protein binds also to a similar polypyrimidine tract present in one of the two strands of a DNA regulatory element of the rat tyrosine aminotransferase gene enhancer. PYBP gene is transcribed ubiquitously as a roughly 2.8 kb RNA which is likely to be subject to an alternative splicing. PYBP is highly homologous to a mouse nuclear protein, as well as to PTB, its human version, which interacts specifically with the pyrimidine tracts of introns. Primary structure information and predicted secondary structure elements of the protein indicate that PYBP contains four sequence repeats. Each of these repeats appears to exhibit the typical RNA recognition motif found in several proteins interacting with RNA or single strand DNA. Finally several hypotheses concerning the biological function of PYBP are presented.

Amino Acid Sequence↗
PubMed1991

Sertoli cell-specific expression of the human transferrin gene. Comparison with the liver-specific expression.

We present a comparative study of the cis- and trans-acting elements governing the expression of the human transferrin (Tf) gene in two tissues, liver and testis, where Tf is expressed at various levels. We have previously identified the elements of the promoter, negative, and enhancer regions involved in the liver-specific expression of the gene. By transfection experiments of primary cultured rat Sertoli cells compared with hepatoma cells, DNase I footprinting, and gel retardation studies, we have analyzed 3.6 kilobase pairs of the Tf regulatory region. The far upstream enhancer functional in Hep3B cells is inactive in Sertoli cells; in the two cell types, different nuclear factors appear to bind to a DNA domain crucial for enhancer activity. Similar negative- and positive-acting elements are present in the distal promoter in both tissues. However different combinations of proximal promoter elements control tissue-specific expression. Liver-specific transcription is governed by the interaction of the Tf-LF1 protein and a C/EBP-related factor with the -125 to -45 region. In Sertoli cells, a -34 to -18 TATA box-binding factor is sufficient to initiate basal-level transcription. Efficient expression is achieved by the association of two factors binding either to the (-82, -1) or to the (-153, -52) region. The addition of a third adjacent element decreases the promoter activity, suggesting that the balance of three factors binding to the proximal sites regulates testis-specific expression.

Animals↗
PubMed1991

The enhancer of the human transferrin gene is organized in two structural and functional domains.

We previously identified a 300-base pair long enhancer, located 3.6 kilobases upstream of the cap site of the human transferrin gene. A 5' deletion up to position 86 of the enhancer resulted in complete loss of the enhancer activity. Here we show by competition footprint analysis, gel retardation assays, and transient expression studies in hepatoma and HeLa cells that the enhancer is composed of two distinct structural and functional domains, A (nucleotides 1-86) and B (nucleotides 87-291). Each domain is a proto-enhancer of a different type. Domain A is a proto-enhancer that, when multimerized, is able by itself to stimulate transcription from the heterologous SV40 promoter, both in Hep3B and HeLa cells. It contains the octanucleotide TGTTTGCT sequence and is the binding site of two liver-specific nuclear factors and of a different HeLa nuclear factor. Domain B contains four binding sites interacting with several liver nuclear proteins. In order to bind, any of these proteins requires the presence of all the others. This domain is able to block the activity of a downstream negative element, but it has no enhancer activity by itself. In the presence of the transferrin promoter, full enhancer activity requires the association of the two domains A and B.

Base Sequence↗
PubMed1990

The binding site for the liver-specific transcription factor Tf-LF1 and the TATA box of the human transferrin gene promoter are the only elements necessary to direct liver-specific transcription in vitro.

We have studied the liver-specific transcriptional activity of the human transferrin gene promoter. Results of competition experiments, site-directed mutagenesis, and 5' deletion analysis have demonstrated that a TATA box and a binding site for the liver-specific protein Tf-LF1 are the only elements needed to direct hepatic-specific transcription in vitro. Thus, Tf-LF1 behaves as other previously described proteins, HNF-1, DBP and LF-A1, in that it is sufficient to confer liver-specific transcriptional activity to a promoter in vitro. This results contrast with observations made in transient expression experiments, in which Tf-LF1 binding alone cannot direct hepatic-specific expression, and the binding of at least one more protein, similar to C/EBP, is needed. Thus, as described for other hepatic genes, the number of elements necessary to confer tissue specificity is different in vivo and in vitro.

Base Sequence↗
PubMed1989

Cell type-specific expression of the human transferrin gene. Role of promoter, negative, and enhancer elements.

Transferrin (Tf), the iron transport protein of vertebrate serum, is mainly synthesized in the liver. cis-Acting DNA elements required for liver-specific expression of the human Tf gene were identified by transient and stable expression assays in human hepatoma (HepG2 and Hep3B) and epithelial carcinoma (HeLa) cell lines. Deletion analysis of the 5' DNA sequences of the gene have defined four functionally different regions: (a) A cell type-specific promoter located between positions -125 and -45 which interacts with two nuclear factors and is sufficient for liver-specific expression. (b) A distal promoter region from -620 to -125 base pairs containing positive and negative cis-acting elements which regulate the promoter activity. (c) A negative-acting region between -1.0 and -0.6 kilobase pairs which down-regulates transcription from the Tf promoter. (d) An enhancer located between -4.0 and -3.3 kilobase pairs which is more active in hepatoma than in HeLa cells. Thus, Tf gene expression is modulated by a combination of multiple positive and negative cis-acting elements. The expression results are discussed with respect to our previous description of the trans-acting factors interacting with the proximal and distal promoter regions.

Base Sequence↗
PubMed1989

Interaction of DNA-binding proteins with the tissue-specific human apolipoprotein-AII enhancer.

The identification of the binding sites for liver nuclear proteins present in the enhancer that control the cell specific transcription of the human apolipoprotein AII gene is reported. Five adjacent binding sites (motifs I to V) were identified. The motifs III, IV and V can be occupied differently by liver or HeLa nuclear proteins. Two hypersensitive zones (between motifs II-III and IV-V) are present only when liver nuclear extracts were tested. A first characterization of the factors reveal that motif IV interacts with the hepatic transcription factors Tf-LF1 (29) and LF-A1 (28, 30). A CCAAT binding protein, different from CTF/NF1, appears to bind to the motif II. The different binding sites share specific DNA sequences principally with 5' regulatory regions of other apolipoprotein genes.

Apolipoprotein A-II↗
PubMed1989

Different liver nuclear proteins binds to similar DNA sequences in the 5' flanking regions of three hepatic genes.

The proximal promoter region of the human transferrin gene contains an hepatocyte-specific cis-element (PRI, nucleotides -76 to -51) whose DNA sequence is homologous to a sequence (nucleotides -89 to -68) present in the transcriptionally essential 5' region of the human antithrombin III gene and to another hepatocyte-specific sequence (A domain) of the human alpha 1-antitrypsin gene promoter. The results reported here lead to the conclusion that the liver trans-acting factor Tf-LF1, binding to the transferrin PRI cis-element interacts with the homologous antithrombin III region, but is different from the transcription factor LF-A1 interacting with the A domain of the alpha 1-antitrypsin promoter. The distal region DRI (nucleotides -480 to -454) of the human transferrin gene promoter presents in its core the same 10 nucleotide-long sequence as the PRI cis-element. We have previously shown that the liver protein Tf-LF2, binding to the DRI element is different from the Tf-LF1 trans-acting factor. In this paper we also show that Tf-LF2 is different from the transcription factor LF-A1 interacting with the alpha 1-antitrypsin promoter. The results allow us to conclude that at least three distinct liver nuclear proteins bind to different subsets of 5' DNA regions containing similar sequences. These sequences are present in genes expressed essentially in liver.

Antithrombin III↗
PubMed1989

The 5' region of Tacaribe virus L RNA encodes a protein with a potential metal binding domain.

We have just completed the Tacaribe arenavirus (TV) genome structure by sequencing the 5' region of the L RNA. Analysis of the sequence has indicated the existence of an open reading frame (ORF) in the viral sense RNA encoding a 95 amino acid polypeptide. The first in phase AUG codon is in positions 70-72 from the 5' end of the viral RNA surrounded by a sequence favorable for the initiation of protein synthesis. The ORF ends at positions 355-357. The predicted polypeptide (P11) contains a cysteine-rich sequence bearing a remarkable similarity to the "zinc finger" sequences found in a number of proteins. We have recently reported that the 3' region of the TV L RNA encodes a polypeptide comprising 2210 amino acids in the viral-complementary sequence. This latter gene, i.e., the L gene, terminates at positions 442-440 from the 5' end of the viral RNA. The two genes encoded by the L RNA (L and P11) are in opposite strands of the RNA in sequences that do not overlap, but are separated by a noncoding intergenic region of 82 nucleotides. The nucleotide sequence of the intergenic region leads to the prediction of a strong secondary structure.

Amino Acid Sequence↗
PubMed1989

Tacaribe virus L gene encodes a protein of 2210 amino acid residues.

The nucleotide sequence of Tacaribe virus (TV) L gene was obtained from two sets of overlapping cDNA clones constructed by walking along the virus L RNA using two successive synthetic DNA primers. Analysis of the sequence indicated the existence of a unique long open reading frame in the viral complementary strand. The first in-phase AUG codon is in positions 31-33 from the 5' end of the viral complementary L RNA surrounded by a sequence favorable for initiation of protein synthesis. The open reading frame ends at positions 6661-6663. The predicted TV L protein is a 2210 amino acid long polypeptide with an estimated molecular weight of 251,942. Comparison of the amino acid sequence of TV L protein with peptide sequences predicted from L-derived cDNA clones of lymphocytic choriomeningitis virus shows an overall 42% of homology.

Amino Acid Sequence↗
PubMed1989

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes.

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.

Antithrombin III↗
PubMed1988

Interactions of DNA-binding proteins with the 5' region of the human transferrin gene.

We have established by transient expression experiments that the 620 base pairs upstream of the cap site of the human transferrin gene contain the information necessary for efficient expression of the gene in hepatoma cells HepG2 or Hep3B but not in HeLa cells. DNase I footprint analysis reveals that at least five distinct factors present in human or rat liver nuclear extracts interact with different sites of this region. One of these factors, binding to nucleotides -193 to -162, is closely related to or identical with the eukaryotic factor CCAAT-binding transcription factor/nuclear factor I; another one, binding to nucleotides -103 to -83 seems to be related to the CCAAT-binding protein. The binding sites of two other factors, not recognized by HeLa nuclear proteins, each contain an identical 10-nucleotide-long sequence (5' TCTTTGACCT 3') in reverse orientation, separated by 400 base pairs. Results of gel retardation assays, cross-competition experiments, and heat inactivation strongly suggest that the proteins binding to these sites are different. One of these sequences and the binding site of the CCAAT-binding protein related factor are located in the region between nucleotides -119 and -45. We have shown by transient expression experiments with 3' deleted vectors that this region is functionally essential for human transferrin gene expression.

Base Sequence↗
PubMed1988

Methionine biosynthesis in Enterobacteriaceae: biochemical, regulatory, and evolutionary aspects.

The genes coding for the enzymes involved in methionine biosynthesis and regulation are scattered on the Escherichia coli chromosome. All of them have been cloned and most have been sequenced. From the information gathered, one can establish the existence (upstream of the structural genes coding for the biosynthetic genes and the regulatory gene) of "methionine boxes" consisting of two or more repeats of an octanucleotide sequence pattern. The comparison of these sequences allows the extraction of a consensus operator sequence. Mutations in these sequences lead to the constitutivity of the vicinal structural gene. The operator sequence is the target of a DNA-binding protein--the methionine aporepressor--which has been obtained in the pure state, for which S-adenosylmethionine acts as the corepressor. Mutations in the corresponding gene lead to the constitutive expression of all the methionine structural genes. The physicochemical properties of the methionine aporepressor are being investigated.

Base Sequence↗