scholarly journals Structure and expression of the mouse Oct2a and Oct2b, two differentially spliced products of the same gene

Development ◽  
1990 ◽  
Vol 109 (2) ◽  
pp. 349-362 ◽  
Author(s):  
A.K. Hatzopoulos ◽  
A.S. Stoykova ◽  
J.R. Erselius ◽  
M. Goulding ◽  
T. Neuman ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Large Family ◽  
Adult Brain ◽  
Cdna Clones ◽  
Reading Frame ◽  
Transcriptional Regulatory ◽  
Octamer Motif ◽  
Additional Exon ◽  
Enhancer Sequences

A large family of tissue-specific nuclear proteins interact with the octamer motif ATTTGCAT, a transcriptional regulatory element found in the promoter and enhancer sequences of many genes. As a step towards elucidating the mechanism of this regulation, cDNA clones of the mouse Oct2 protein were isolated. One, called here Oct2b, encodes a larger variant of the previously described Oct2a proteins. The Oct2b cDNA has an insertion of 74 bp close to the 3′ end which creates an open reading frame distinct from Oct2a. As a result, the Oct2b protein has a carboxy end which is similar to that of the ubiquitous octamer-binding protein Oct1. Analysis of the Oct2 gene shows that Oct2a and Oct2b are differentially spliced products of the same gene. The insertion in the Oct2b cDNA results from the inclusion of an additional exon in the mRNA which would otherwise reside in an intron sequence of the Oct2a transcript. RNA analysis demonstrates that both Oct2a and 2b mRNAs are most abundant in B-cells but they are also expressed in a variety of tissues including brain, intestine, testis, kidney, as well as in embryos. Interestingly, the ratio of Oct2a and 2b varies among tissues. In situ hybridization studies during mouse embryogenesis show that the Oct2 gene is widely expressed in the developing nervous system. In contrast, expression in the adult brain is confined to very specific areas which include the suprachiasmatic and medial mammillary nuclei, hippocampus, olfactory tract and the olfactory bulb. Oct2 proteins are present in both neuronal and oligodendroglial cells, although they are more abundant in glial cells.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

scholarly journals A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes.

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654 ◽  
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters

1990 ◽  
Vol 10 (12) ◽  
pp. 6204-6215
Author(s):  
A Pierani ◽  
A Heguy ◽  
H Fujii ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Regulatory Element ◽  
Immunoglobulin Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Activation Domains ◽  
Cell Extracts ◽  
Octamer Motif ◽  
High Level

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

Spinalin, a new glycine- and histidine-rich protein in spines of Hydra nematocysts

1998 ◽  
Vol 111 (11) ◽  
pp. 1545-1554 ◽  
Author(s):  
A.W. Koch ◽  
T.W. Holstein ◽  
C. Mala ◽  
E. Kurz ◽  
J. Engel ◽  
...  
Keyword(s):  
External Surface ◽  
Insoluble Fraction ◽  
Cdna Clones ◽  
Mature Protein ◽  
Capsule Wall ◽  
The Matrix ◽  
Acidic Region ◽  
Basic Region ◽  
Full Length Clone

Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst's capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors

1993 ◽  
Vol 13 (8) ◽  
pp. 4494-4504
Author(s):  
D Nitsch ◽  
G Schütz
Keyword(s):  
Thymidine Kinase ◽  
Specific Binding ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Sequences

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

Claudin-2 is selectively expressed in proximal nephron in mouse kidney

2001 ◽  
Vol 281 (5) ◽  
pp. F966-F974 ◽  
Author(s):  
Alissa H. Enck ◽  
Urs V. Berger ◽  
Alan S. L. Yu
Keyword(s):  
Tight Junction ◽  
Large Family ◽  
Mouse Kidney ◽  
High Rate ◽  
Structural Components ◽  
Water Reabsorption ◽  
Junction Protein ◽  
Confocal Images ◽  
Leaky Epithelium

First published August 15, 2001; 10.1152/ajprenal.00021.2001.—The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

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