scholarly journals Molecular characterization of a novel transcription factor that controls stromelysin expression.

1995 ◽  
Vol 15 (6) ◽  
pp. 3164-3170 ◽  
Author(s):  
L Sanz ◽  
J Moscat ◽  
M T Diaz-Meco
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Critical Role ◽  
Single Copy ◽  
Binding Activity ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Binding Domains ◽  
3T3 Fibroblasts ◽  
Simplex Virus

Stromelysins, which are the metalloproteinases with the widest substrate specificities, play a critical role in tumor invasion and metastasis. We have previously reported an element (SPRE) of the stromelysin promoter located between nucleotides -1221 and -1203 that is necessary and sufficient for the control of stromelysin gene expression by mitogenic activation, which induces a nuclear activity that binds to this sequence. Using a concatenated probe with several copies of this element to screen a lambda gt11 cDNA expression library from mouse Swiss 3T3 fibroblasts, we report here the molecular cloning of a cDNA coding for a novel protein (SPBP) of 937 amino acids that binds to this element and has several features of a transcription factor, such as a putative leucine zipper region, a nuclear localization signal, and a basic domain with homology to the DNA-binding domains of Fos and Jun. Evidence that SPBP is at least a critical component of the mitogen-induced SPRE nuclear binding activity is presented here. Furthermore, the transfection of an expression plasmid for SPBP transactivates reporter chloramphenicol acetyltransferase plasmids containing either the full-length stromelysin promoter or a single copy of the SPRE cloned upstream of the herpes simplex virus thymidine kinase minimal promoter. Therefore, the results presented here identify a novel transcription factor critically involved in the control of stromelysin expression.

scholarly journals Suppression of E1A-Mediated Transformation by the p50E4F Transcription Factor

1999 ◽  
Vol 19 (7) ◽  
pp. 4739-4749 ◽  
Author(s):  
Elma R. Fernandes ◽  
Robert J. Rooney
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Suppressive Effect ◽  
Culture Conditions ◽  
Binding Activity ◽  
Cellular Transcription Factor ◽  
Dna Binding Activity ◽  
3T3 Fibroblasts ◽  
Induced Apoptosis ◽  
Nih 3T3

ABSTRACT The adenovirus E1A gene can act as an oncogene or a tumor suppressor, with the latter effect generally arising from the induction of apoptosis or the repression of genes that provide oncogenic growth stimuli (e.g., HER-2/c-erbB2/neu) or increased metastatic invasiveness (e.g., metalloproteases). In this study, coexpression of E1A and p50E4F, a cellular transcription factor whose DNA binding activity is stimulated by E1A, suppressed colony formation by NIH 3T3 cells and transformation of primary rat embryo fibroblasts but had no observed effect in the absence of E1A. Domains in p50E4F required for stimulation of the adenovirus E4 promoter were required for the suppressive effect, indicating a transcriptional mechanism. In serum-containing media, retroviral expression of p50E4F in E1A13S/ras-transformed NIH 3T3 fibroblasts had little effect on subconfluent cultures but accelerated a decline in viability after the cultures reached confluence. Cell death occurred by both apoptosis and necrosis, with the predominance of each process determined by culture conditions. In serum-free media, p50E4F accelerated E1A-induced apoptosis. The results suggest that p50E4F sensitizes cells to signals or conditions that cause cell death.

Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP

1991 ◽  
Vol 11 (10) ◽  
pp. 4863-4875
Author(s):  
S V Iyer ◽  
D L Davis ◽  
S N Seal ◽  
J B Burch
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Protein ◽  
Expression Profiles ◽  
Control Element ◽  
Cdna Expression Library ◽  
Leucine Zippers ◽  
Expression Library ◽  
Positive Elements ◽  
Vitellogenin Gene

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.

scholarly journals Proteophosphoglycans of Leishmania mexicana: Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes

1999 ◽  
Vol 344 (3) ◽  
pp. 787-795 ◽  
Author(s):  
Ulrich GÖPFERT ◽  
Nathan GOEHRING ◽  
Christian KLEIN ◽  
Thomas ILG
Keyword(s):  
Mrna Level ◽  
Single Copy ◽  
Gene Replacement ◽  
Mammalian Host ◽  
Cdna Expression Library ◽  
Dna Repeats ◽  
The Novel ◽  
Leishmania Mexicana ◽  
Expression Library ◽  
Gene Encoding

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressedppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

scholarly journals Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

2004 ◽  
Vol 72 (4) ◽  
pp. 2194-2202 ◽  
Author(s):  
Kimberly Campbell ◽  
Vsevolod Popov ◽  
Lynn Soong
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Single Copy ◽  
Interleukin 10 ◽  
Leishmania Amazonensis ◽  
Cdna Expression Library ◽  
Protein Protein Interactions ◽  
Expression Library ◽  
Gene Encoding ◽  
Mucocutaneous Leishmaniasis

ABSTRACT Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ∼3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4+ cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.

scholarly journals Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein

1994 ◽  
Vol 14 (11) ◽  
pp. 7633-7642
Author(s):  
K A Lillycrop ◽  
S J Dawson ◽  
J K Estridge ◽  
T Gerster ◽  
P Matthias ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Critical Role ◽  
Terminal Region ◽  
Early Gene ◽  
Immediate Early ◽  
C Terminus ◽  
N Terminus ◽  
Simplex Virus

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

scholarly journals elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family.

1991 ◽  
Vol 11 (9) ◽  
pp. 4651-4659 ◽  
Author(s):  
J Spieth ◽  
Y H Shim ◽  
K Lea ◽  
R Conrad ◽  
T Blumenthal
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Dna Binding ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Recognition Sequence ◽  
Sequence Element ◽  
C Elegans ◽  
Binding Domains ◽  
Degenerate Oligonucleotide

The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.

elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family

1991 ◽  
Vol 11 (9) ◽  
pp. 4651-4659
Author(s):  
J Spieth ◽  
Y H Shim ◽  
K Lea ◽  
R Conrad ◽  
T Blumenthal
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Dna Binding ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Recognition Sequence ◽  
Sequence Element ◽  
C Elegans ◽  
Binding Domains ◽  
Degenerate Oligonucleotide

The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53

1994 ◽  
Vol 14 (10) ◽  
pp. 7013-7024 ◽  
Author(s):  
H Xiao ◽  
A Pearson ◽  
B Coulombe ◽  
R Truant ◽  
S Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Factor B ◽  
Activation Domains ◽  
Binding Domains ◽  
General Transcription Factors ◽  
Transcriptional Activation Domains ◽  
Simplex Virus

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus

Parasitology ◽  
1999 ◽  
Vol 119 (4) ◽  
pp. 405-412 ◽  
Author(s):  
P. J. SKUCE ◽  
D. L. REDMOND ◽  
S. LIDDELL ◽  
E. M. STEWART ◽  
G. F. J. NEWLANDS ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Southern Blot Analysis ◽  
Single Copy ◽  
Blood Feeding ◽  
Cdna Expression Library ◽  
Cysteine Proteinases ◽  
Rt Pcr ◽  
Expression Library ◽  
Developmentally Regulated

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a ‘proteinase-enriched’ Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT–PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

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