bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control.
Authors: Levy D E , Kessler D S , Pine R , Reich N , Darnell J E
Published: June 2007

Human alpha- and beta-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription.
Dwarf mice produced by genetic ablation of growth hormone-expressing cells.
Authors: Behringer R R , Mathews L S , Palmiter R D , Brinster R L
Published: June 2007

Fusion of the 310 bp located 5' of the rat growth hormone (GH) gene to the human GH structural gene resulted in somatotrope-specific expression in transgenic mice. Human GH transcripts were detected only in pituitaries of these mice, and immunocytochemical analyses revealed that this expression was limited to GH-expressing cell types. The rat GH 5' sequences were then used to direct the expression of diphtheria toxin to the GH-expressing cells of transgenic mice. A line of mice was established which lacks detectable levels of circulating GH. This deficiency resulted in dwarfism; transgenic animals grew only to half the size of nontransgenic littermates. Nearly all somatotropes were absent, as shown by GH immunostaining in the transgenic pituitaries. Prolactin (PRL)-producing lactotropes, thought to share a common cellular origin with somatotropes, were also reduced in numbers. A model for the lineal relationships between GH- and PRL-synthesizing cells is proposed.
Identification of the gene coding for the Endo B murine cytokeratin and its methylated, stable inactive state in mouse nonepithelial cells.
Authors: Oshima R G , Trevor K , Shevinsky L H , Ryder O A , Ceceña G
Published: June 2007

The Endo B type-I keratin intermediate filament protein is first expressed at the 4- to 8-cell stage of mouse development. In the adult, its expression is restricted to a variety of simple epithelial cell types. To investigate the mechanisms responsible for the restricted expression of Endo B, the gene coding for Endo B has been identified from among the five different Endo B genes found in the mouse genome by Southern hybridization analysis and cloning all or part of four of the genes. Nuclear run-on experiments demonstrate that Endo B expression is regulated at the level of transcription. The 5' end of the active gene, designated Endo beta 1, was found to be highly methylated and in a relatively nuclease-resistant chromatin conformation in fibroblasts and myoblasts that do not express Endo B, but undermethylated and relatively sensitive to nuclease digestion in endodermal cells or F9 embryonal carcinoma cells. The inactive state of the Endo B beta 1 gene in fibroblast appears to be very stable, because somatic cell hybrids formed by the fusion of HeLa cells, which express the homologous human protein, keratin 18, and mouse fibroblasts, continue to express keratin 18 but do not activate Endo B expression. Similarly, the fusion of mouse endodermal cells and fibroblasts results in hybrids that do not extinguish Endo B expression. These results suggest that Endo B transcription is limited by two different mechanisms. In somatic cells such as fibroblasts or myoblasts, expression may be restricted by methylation and a stable, nonpermissive transcriptional state. However, in embryonal carcinoma cells, the Endo B beta 1 gene is undermethylated and in a relatively nuclease-sensitive conformation, but it is restricted by an additional, negative regulatory mechanism.
Deletion of nonconserved helices near the 3' end of the rRNA intron of Tetrahymena thermophila alters self-splicing but not core catalytic activity.
Authors: Barfod E T , Cech T R
Published: June 2007

The self-splicing rRNA intron of Tetrahymena thermophila contains two stem-loop structures (P9.1 and P9.2) near its 3' end that are not conserved among group I introns. As a step toward deriving the smallest active self-splicing RNA, 78 nucleotides encompassing P9.1 and P9.2 have been deleted. This deletion has no effect on the core catalytic activity of the intron, as judged by its ability to catalyze poly(C) polymerization and other related reactions. In contrast, reactions at the 3' splice site of the rRNA precursor--exon ligation and intermolecular exon ligation--take place with reduced efficiency, and exon ligation becomes rate-limiting for self-splicing. Moreover, intermolecular exon ligation with pentaribocytidylic acid is inaccurate, occurring primarily at a cryptic site in the 3' exon. A deletion of 79 nucleotides that disrupts P9, as well as removing P9.1 and P9.2, has more severe effects on both the first and second steps of splicing. P9, a conserved helix at the 5' edge of the deletion point, can form stable alternative structures in the deletion mutants. This aberrant folding may be responsible for the reduced activity and accuracy of reactions with mutant precursors. Analysis of the cryptic site suggests that choice of the 3' splice site may not only depend on sequence but also on proximity to P9. In the course of these studies, evidence has been obtained for an alternative 5' exon-binding site distinct from the normal site in the internal guide sequence.
Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein.
Authors: Tsay Y F , Thompson J R , Rotenberg M O , Larkin J C , Woolford J L
Published: June 2007

We have investigated the mechanisms whereby equimolar quantities of ribosomal proteins accumulate and assemble into ribosomes of the yeast Saccharomyces cerevisiae. Extra copies of the cry1 or RPL16 genes encoding ribosomal proteins rp59 or L16 were introduced into yeast by transformation. Excess cry1 or RPL16 mRNA accumulated in polyribosomes in these cells and was translated at wild-type rates into rp59 or L16 proteins. These excess proteins were degraded until their levels reached those of other ribosomal proteins. Identical results were obtained when the transcription of RPL16A was rapidly induced using GAL1-RPL16A promoter fusions, including a construct in which the entire RPL16A 5'-noncoding region was replaced with the GAL1 leader sequence. Our results indicate that posttranscriptional expression of the cry1 and RPL16 genes is regulated by turnover of excess proteins rather than autogenous regulation of mRNA splicing or translation. The turnover of excess rp59 or L16 is not affected directly by mutations that inactivate vacuolar hydrolases.
Expression of nodule-specific genes in alfalfa root nodules blocked at an early stage of development.
Authors: Dickstein R , Bisseling T , Reinhold V N , Ausubel F M
Published: June 2007

To help dissect the molecular basis of the Rhizobium-legume symbiosis, we used in vitro translation and Northern blot analysis of nodule RNA to examine alfalfa-specific genes (nodulins) expressed in two types of developmentally defective root nodules elicited by Rhizobium meliloti. Fix- nodules were elicited by R. meliloti nif mutants; these nodules were invaded by rhizobia and contained differentiated bacteroids. 'Empty' nodules were elicited by R. meliloti exo and ndv mutants and by Agrobacterium tumefaciens strains carrying the R. meliloti nod genes; these nodules contained a nodule meristem but lacked infection threads, intracellular bacteria, and bacteroids. Fix- nodules contained a spectrum of nodulins similar to wild-type nodules. In contrast, only two nodulins, Nms-30 and a nodulin homologous to ENOD2 of soybean, were detected in empty nodules. Although R. meliloti ndv and exo mutants elicited nodules with the same defective phenotype, ndv and exo mutants (except for exoC mutants) had distinct biochemical phenotypes. R. meliloti ndvA and ndvB mutants were deficient in cyclic glucan production but not the acidic exopolysaccharide; the converse was true for exoA, exoB, and exoF mutants. exoC mutants were defective in both exopolysaccharide and cyclic glucan biosynthesis. Our results support the model that the R. meliloti nod genes produce a signal that results in nodule meristem induction. Both the exopolysaccharide and cyclic glucan, however, appear to act at the next step in the developmental process and are involved in the production of a signal (or structure) that allows infection thread formation and invasion of the nodule.
The positive and negative transcriptional regulation of the Drosophila Gapdh-2 gene.
Authors: Sun X H , Lis J T , Wu R
Published: June 2007

One of the genes encoding glyceraldehyde 3-phosphate dehydrogenase of Drosophila melanogaster, Gapdh-2, is expressed in all cell types examined, but its level of expression is regulated developmentally. Here we report the analysis of the regulatory sequences for the transcription of Gapdh-2. We have generated Gapdh-2-LacZ fusion genes in which the 5'-flanking sequence of Gapdh-2 has been mutated. Examination of the expression of these fusion genes, which have been introduced by transfection into the Schneider II cell line and by germ-line transformation into flies, led to the identification of two distinct regulatory regions, URS-1 and URS-2, within the first 145 bp of the 5'-flanking sequence of Gapdh-2. URS-1 activates transcription throughout the development of Drosophila. However, URS-2 exhibits a dual function during the development. It clearly represses transcription in Schneider II cells, and perhaps also in mid-stage embryos. In contrast, it activates transcription in larvae and adult flies. Thus, URS-2 plays a key role in the developmental regulation of Gapdh-2. Additionally, the efficient transcription of Gapdh-2 in larval and adult stages appears to depend on a synergistic function of URS-1 and URS-2.
Transcription by RNA polymerase II induces changes of DNA topology in yeast.
Authors: Osborne B I , Guarente L
Published: June 2007

We show that induction of transcription of a CYC1-lacZ fusion gene, borne on a yeast plasmid, causes an increase in negative superhelicity of approximately five turns. This increase is abolished by deletion of either essential element of the CYC1 promoter, the upstream activation site (UAS), or the TATA boxes. Several experiments indicate that the size of the increase is proportional to the size of the transcribed region. First, an internal deletion removing half of the CYC1-lacZ transcribed region results in a plasmid whose negative superhelicity on induction is intermediate between promoter-deletion plasmids and the parental plasmid. Second, plasmids bearing insertions of a fragment containing the putative CYC1 terminator into the CYC1-lacZ fusion gene have relative negative superhelicities proportional to the length of the truncated fusion transcripts generated. A plausible model explaining these observations is that local unwinding of the double helix by transcribing RNA polymerase generates positively supercoiled DNA, which is subsequently relaxed by a topoisomerase.
Xenopus endo B is a keratin preferentially expressed in the embryonic notochord.
Authors: LaFlamme S E , Jamrich M , Richter K , Sargent T D , Dawid I B
Published: June 2007

Screening of a cDNA library from neurula stage Xenopus laevis for notochord-specific sequences led to the isolation of a cDNA clone, XK endo B (Xenopus keratin endo B), which encodes a nonepidermal type I keratin. In situ and Northern blot hybridizations indicate that expression of XK endo B RNA is concentrated in the notochord, whereas expression in the endoderm is 5-10 times lower. XK endo B mRNA is present in the oocyte and increases from late gastrula. Accumulation peaks by late neurula and is greatly reduced by the tadpole stage; in the adult, a low level of XK endo B RNA is present in the liver. XK endo B shows sequence homology to mouse endo B; genomic Southern blots show that XK endo B is the most similar sequence to mouse endo B in the Xenopus genome, and vice versa, indicating that XK endo B and mouse endo B are homologs. The use of endo B as a marker and the germ layer derivation of the notochord are discussed in light of these results.
Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress.
Authors: Kusukawa N , Yura T
Published: June 2007

An Escherichia coli mutant lacking the heat shock sigma-factor (sigma 32) is defective in transcription from heat shock promoters and cannot grow at temperatures above 20 degrees C. To assess physiological roles of sigma 32 and heat shock proteins, we isolated and characterized a set of temperature-resistant revertants from this deletion (delta rpoH) mutant. Most of them were found to carry a DNA insertion in the groE upstream region, resulting in high-level synthesis of major heat shock proteins GroE (GroES and GroEL). The levels of GroE produced varied in different revertants and correlated well with the maximum permissive temperatures; the highest GroE producers (approximately 10% of total protein) grew up to 40 degrees C but not at 42 degrees C. An additional mutation causing hyperproduction of DnaK (hsp70 homolog) was required for growth at 42 degrees C. Such effects of GroE and DnaK on the sigma 32-deletion strains were also confirmed by using multicopy plasmids carrying groE or dnaK. Thus, GroE plays a key protective role in supporting growth at normal physiological temperatures (20-40 degrees C), whereas high levels of DnaK are required primarily at higher temperature.
Expression, function, and regulation of the hairy segmentation protein in the Drosophila embryo.
Authors: Carroll S B , Laughon A , Thalley B S
Published: June 2007

The Drosophila pair-rule segmentation gene, hairy, is required for the proper development of alternate embryonic segment primordia and the normal spatial expression of another pair-rule gene, fushi tarazu (ftz). We demonstrate that the product of the hairy gene is localized to the nuclei of cells in eight distinct regions of the early embryo. The major stripes of protein accumulation comprise the posterior half and the adjacent anterior portion of alternate segment primordia; the hairy pattern overlaps transiently with ftz expression in a narrow band of cells. Unlike other pair-rule products that have been studied, hairy is not expressed in the embryonic nervous system. The spatial distribution of the nuclear hairy protein and the pattern of ftz expression in embryos lacking hairy function suggests that hairy plays a direct role in repressing ftz gene expression. The pattern of hairy expression in gap gene mutants that affect ftz expression indicates that the gap genes largely act indirectly upon ftz through their effects on hairy.
Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore.
Authors: Zheng L B , Donovan W P , Fitz-James P C , Losick R
Published: June 2007

Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is a morphogenic protein required in the assembly of the outer coat. The nucleotide sequence of cotE reveals an open reading frame capable of encoding a 181-residue-long polypeptide of 21 kD. A cotE mutant was created by replacing the chromosomal gene, which was located at 145 degrees on the chromosome, with an in vitro constructed, deletion-mutated gene. The resulting cotE mutant formed normal-looking (optically refractile) spores that were heat resistant but were sensitive to lysozyme and somewhat impaired in germination. Ultrastructural analysis indicated that the mutant spores lacked the electron-dense outer layer of the coat but retained a normal-looking inner coat. The mutant spores were pleiotropically deficient in several coat proteins, including the product of cotE and the products of previously cloned cot genes A-C. Based on experiments in which expression of the cotA and cotC genes was found to be unimpaired in cotE mutant cells, we infer that the cotE gene product is involved in the assembly of the products of cotA-cotC, and certain other proteins into the electron-dense outer layer of the coat.
Phase variation in Salmonella: analysis of Hin recombinase and hix recombination site interaction in vivo.
Authors: Hughes K T , Youderian P , Simon M I
Published: June 2007

The bacteriophage P22-based challenge phase selection was used to characterize the binding of Salmonella Hin recombinase to the wild-type hixL and hixR recombination sites, as well as to mutant and synthetic hix sequences in vivo. Hin recombinase binds to the hixL or hixR recombination sites and represses transcription from an upstream promoter in the challenge phage system. Hin-mediated repression results from Hin associating into multimers either prior to binding or during the binding process at the hix operator sites (cooperativity). The ability of Hin multimers to repress transcription is eliminated when the hix 13-bp half-sites are rotated to opposite sides of the DNA helix by inserting 4 bp between them. Insertion of 1 bp between half-sites reduces overall repression. Hin also binds one of the hixL half-sites to repress transcription, but only when high levels of Hin protein are present in the cell. Mutations have been identified in the hix sites that impair Hin binding. Five of the 26 bp in the hix sites are critical; sites with base-pair substitutions at these five positions show greatly reduced binding. Three additional base pairs make minor contributions to binding. These results are consistent with the results of binding studies between Hin and the hix sites in vitro.
Configuration of the alpha-fetoprotein regulatory domain during development.
Authors: Godbout R , Tilghman S M
Published: June 2007

The transcription of the mouse alpha-fetoprotein gene in the fetal liver, gut, and yolk sac is under the control of at least four regulatory sequences, three 5' distal enhancers, and a proximal promoter region. In transgenic mice, the three enhancers exhibited distinct tissue preferences, with all three active in the visceral endoderm of the yolk sac, two in the fetal liver, and one in the fetal gut. To ask whether the enhancers are differentially utilized by the endogenous gene in the three tissues in vivo, we examined their differential sensitivity to the endonuclease DNase I. The experiments indicated that two of the three enhancers exhibited sensitivity to DNase I in all three fetal tissues, as well as in the adult liver, where the gene is transcriptionally repressed. The third enhancer, which exhibited the weakest activity in transgenic mice, was the least sensitive to DNase I in fetal liver and yolk sac and insensitive in the fetal gut. The major changes in the chromatin structure of the gene during postnatal development occurred within the proximal promoter region. The major DNase I cleavage site shifted from a position at 120 nucleotides upstream of the transcriptional start site to the start site itself. Two new sites, at 300 nucleotides upstream of the start site and 1.5 kb within the structural gene, were observed. These results suggest that the distal regulatory regions have the capability to retain biological activity throughout development, and that the primary repression of transcription of the gene proceeds through elements proximal to the promoter of the gene.
EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.
Authors: Cortes P , Buckbinder L , Leza M A , Rak N , Hearing P , Merino A , Reinberg D
Published: June 2007

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.
Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter.
Authors: Watt F , Molloy P L
Published: June 2007

Cytosine methylation within CpG dinucleotides has been implicated in the regulation of gene expression in vertebrates and, in some cases, has been shown to be causative in repression of transcription. We have examined whether methylation of CpG dinucleotides located within the binding site for a specific transcription factor, MLTF or USF, affects its binding to DNA. This HeLa cell factor binds to the adenovirus major late promoter (AdMLP), as well as endogenous cellular genes, and stimulates transcription in an in vitro assay. Synthetic oligonucleotides in which 5-methylcytosine replaces cytosine at specific sites were used to generate duplex DNAs, and the formation of complexes of these oligomers with MLTF was studied using a gel retardation assay. Methylation at a CpG site centrally located within the binding site strongly inhibited complex formation, whereas methylation at a site 6 bases away had no demonstrable effect. Methylation at the central site was also shown to inhibit specific transcription in vitro from the AdMLP. Methylation at the central site on only one strand caused a partial inhibition of binding, the effect being greater when the noncoding strand was methylated. The results indicate that in some cases, site-specific methylation may inhibit gene expression directly by blocking binding to DNA of factors required for optimal transcription. Along with other recent findings, they suggest an interplay between DNA methylation and transcription factors in the regulation of gene expression.
Lens-specific expression of recombinant ricin induces developmental defects in the eyes of transgenic mice.
Authors: Landel C P , Zhao J , Bok D , Evans G A
Published: June 2007

An expression system for cell lineage ablation in transgenic mice was constructed in which a modified form of the A subunit of ricin, a toxic lectin produced by the castor bean Ricinus communis, can be expressed under the direction of tissue-specific regulatory signals. A chimeric gene was formed by fusing the promoter and 5'-flanking sequences of the lens-specific mouse alpha A-crystallin gene with a modified ricin A cDNA, and this construction was integrated into the germ line of transgenic mice. These animals develop profound microphthalmia with severe developmental defects of the eye, relating primarily to the disorganization and death of cells forming the lens. In addition, this defect is associated with several abnormalities, including eye size, folding of the retina, and ectopic lens material in other regions of the eye. The phenotype of this engineered developmental mutation suggests that the normal development of alpha A-crystallin-producing lens fiber cells is essential for the proper growth, organization, and orientation of optic structures.
Isolation of the Drosophila segmentation gene runt and analysis of its expression during embryogenesis.
Authors: Gergen J P , Butler B A
Published: June 2007

runt is one of the genes required for establishment of the segmented body pattern of the Drosophila embryo. We have isolated DNA sequences containing this gene using P-element transposon tagging. Southern blot analyses of six different DNA rearrangements that are associated with runt mutations revealed a minimal region of 8.5-kb of DNA that was important for function. In germ line transformation experiments, a 14.5-kb segment of DNA that spanned this minimal region provided significant, although not full, levels of runt activity. The runt gene encoded a 2.6-kb poly(A)+ RNA that underwent a series of dynamic changes in its spatial and temporal patterns of accumulation during embryogenesis. The runt RNA was most abundant at the blastoderm stage when it showed the seven stripes of expression characteristic of other Drosophila pair-rule genes.
Spatial regulation of engrailed expression in the Drosophila embryo.
Authors: Weir M P , Edgar B A , Kornberg T , Schubiger G
Published: June 2007

Novel patterns of engrailed RNA were observed in early Drosophila embryos injected with cycloheximide, an inhibitor of protein synthesis. From these patterns, we infer that there are several superimposed systems of spatial regulation which in combination localize engrailed expression in the embryo. Activation of engrailed transcription progresses with an anterior-to-posterior polarity. Superimposed are systems of negative regulation that repress expression in the anterior 30% of the embryo and in the interbands between stripes. We suggest that products of known segmentation genes are the repressors that suppress engrailed expression in interbands.
The segmentation gene Krüppel of Drosophila melanogaster has homeotic properties.
Authors: Harbecke R , Janning W
Published: June 2007

In Drosophila hindgut, Malpighian tubules and posterior midgut develop from the most posterior region of the blastoderm. One of the genes that influences the differentiation of the Malpighian tubules is Krüppel (Kr), a segmentation gene of the gap class. Kr homozygous embryos lack thoracic and abdominal segments and, depending on the allele, develop nearly normal Malpighian tubules or do not differentiate them at all. In the wild type, injection of horseradish peroxidase (HRP) into cells of the early gastrula at various posterior positions results in labeling of hindgut (93%), Malpighian tubules (46%), and posterior midgut (20%). Malpighian tubules were labeled only in combination with hindgut. In Kr1 homozygous embryos that lack Malpighian tubules, the label was restricted to hindgut (84%) and posterior midgut (24%). Because we could not find significant cell death in the posterior region of Kr1 embryos, we counted the cell nuclei in the hindguts of wild-type and mutant embryos. The results show that the hindgut in Kr1 embryos contains those cells that would differentiate into Malpighian tubules in wild type. Therefore, we conclude that the Krüppel gene exhibits a homeotic function in addition to its role as a segmentation gene and is involved in separating hindgut and Malpighian tubule cells and in the elongation process as well.
Widespread expression of human alpha 1-antitrypsin in transgenic mice revealed by in situ hybridization.
Authors: Koopman P , Povey S , Lovell-Badge R H
Published: June 2007

In situ hybridization is a powerful means of identifying sites of gene expression. We used this technique to examine the spatial and developmental control of transcription of the human alpha 1-antitrypsin (alpha 1 AT) gene in transgenic mice carrying this gene and extensive 5'- and 3'-flanking sequences. In addition to expression in yolk sac and liver, human alpha 1AT RNA was detected in gut, stomach, pancreas, nasal epithelium, pharynx, bronchi, spinal ganglia, and ossifying cartilage of transgenic fetuses at 14.5 days post coitum (dpc). In transgenic adults, expression was no longer found in the pancreas but was found in the kidney and salivary gland. In each tissue, expression was confined to a specific cell population. This pattern of alpha 1AT expression was found to correlate with that seen in several fetal and adult human tissues. These results suggest a wider role of alpha 1AT in human physiology and development than previously suspected, and they demonstrated the potential value of this approach in delineating the physiological role of human proteins. Expression of the endogenous alpha 1AT gene in mice was confined to a limited, but overlapping, set of tissues, suggesting that the cis-acting DNA sequences that regulate the expression of the human and mouse genes interact differently with transcription factors present in mouse cells.
A new family of mouse homeo box-containing genes: molecular structure, chromosomal location, and developmental expression of Hox-7.1.
Authors: Hill R E , Jones P F , Rees A R , Sime C M , Justice M J , Copeland N G , Jenkins N A , Graham E , Davidson D R
Published: June 2007

Two families of homeo box-containing genes have been identified in mammals to date, the Antennapedia- and engrailed-like homeo boxes, based on the sequence similarity to those from Drosophila. Here, we report the isolation of a homeo box-containing gene that belongs to a new family of which there are at least three related genes in the mouse genome. The homeo box of this new gene shows remarkable similarity to the Drosophila Msh homeo box that we designate as the prototype for this family. The gene maps to the proximal end of mouse chromosome 5 and does not cosegregate with any known homeo box-containing gene. We designate this locus Hox-7.1. In situ hybridizations to mouse embryos at different stages show a unique pattern of expression, as compared to other homeo box-containing genes described thus far. Hox-7.1 transcripts are detected in 9.5-day-old embryos in the neural crest, developing limb bud, and visceral arches. Later, this gene is expressed in regions of the face that are derived from neural crest and in the interdigital mesenchymal tissues in both the fore- and hindlimbs.
Functional analysis of the papilloma virus E2 trans-activator in Saccharomyces cerevisiae.
Authors: Lambert P F , Dostatni N , McBride A A , Yaniv M , Howley P M , Arcangioli B
Published: June 2007

The papilloma virus E2 transcriptional trans-activator is representative of a class of transcriptional modulators that activate transcription through direct binding to cis-acting DNA sequences. In this study we measured the capacity for this mammalian virus factor to function in Saccharomyces cerevisiae. When expressed in the yeast, the bovine papilloma virus E2 trans-activator could stimulate transcription from a yeast promoter having E2 DNA-binding sites present in cis. Whereas a single E2 DNA-binding site was sufficient for trans-activation, a strong cooperative effect was observed with two E2 DNA-binding sites. The level of trans-activation was dependent on the position of the E2 DNA-binding sites in relation to the yeast promoter, with the maximal effect demonstrated when the binding sites were positioned upstream. Deleted E2 proteins, lacking part of the trans-activation or DNA-binding domains, failed to activate transcription in yeast, similar to their behavior in mammalian cells. Replacement of the amino-terminal region of the E2 trans-activation domain with a synthetic amphipathic helix partially restored the trans-activation function; however, it did not result in a molecule that exhibited cooperativity between neighboring E2 DNA-binding sites.
Promoter occlusion prevents transcription of adenovirus polypeptide IX mRNA until after DNA replication.
Authors: Vales L D , Darnell J E
Published: June 2007

The left end of the adenovirus genome is arranged such that the polypeptide IX gene is 'buried' (entirely contained) within the E1B transcription unit. The E1B gene is transcribed actively early in infection while, in contrast, IX gene transcription only occurs after DNA replication. Using recombinant plasmid constructs and recombinant viruses, we have found that the nested arrangement of the IX gene prevents its transcription. The experiments show that E1B transcription across the IX promoter inhibits IX gene expression early in infection, and yet, the 21-kD E1B protein activates the IX gene. IX mRNA synthesis occurs in the absence of DNA replication when the E1A gene and E1B promoter are absent, but only when the 21-kD E1B protein is present in trans. Our results indicate that during the adenovirus infectious cycle, the only templates on which IX transcription can be activated are newly replicated templates not committed to E1B transcription. This situation may be a model for genes that are activated specifically at the time of replication.
Two transcripts from the rotund region of Drosophila show similar positional specificities in imaginal disc tissues.
Authors: Agnel M , Kerridge S , Vola C , Griffin-Shea R
Published: June 2007

The rotund (rn) mutation in Drosophila is unique in that its phenotype is limited to the deletion of specific distal parts, though not the extremities, of all adult appendages. We have cloned the rn gene, located at cytogenetic position 84D3,4, by chromosomal walking. The functional rn unit, defined genetically by the localization of 13 noncomplementing rn alleles, covers approximately 50 kb of DNA. Despite different developmental profiles, two transcript size classes (1.7 and 5.3 kb) from this region show an indistinguishable pattern of spatial expression in the imaginal discs at the white pupa stage. There is a high correlation between the specificity of the mutant phenotype and the accumulation of transcripts in the presumptive distal regions of the cuticle-forming epithelial cells of the affected discs; it is, in fact, the first gene reported whose expression is localized with respect to the proximo distal-forming axis. For both transcripts, we have also found, uniquely in the wing disc, expression limited to the anterior region of the mesodermally derived epithelial cells, which contribute to the muscles of the thorax.
The limb deformity gene is required for apical ectodermal ridge differentiation and anteroposterior limb pattern formation.
Authors: Zeller R , Jackson-Grusby L , Leder P
Published: June 2007

To gain insight into the role of the limb deformity (ld) gene in limb morphogenesis, we examined the morphologic details of early embryonic limb formation in the mutant ld/ld mouse. Initial morphological differences between wild-type and homozygous ld embryos are apparent during early gestational day 10, a time period during which anteroposterior limb morphogenesis occurs. As a result of a shortened anteroposterior axis, the mutant limb bud appears more pointed than its wild-type counterpart. In addition, the apical ectodermal ridge (AER), a structure crucial to both proximodistal and anteroposterior limb development, fails to differentiate properly in mutant ld embryos. Consistent with these observations, molecular analysis of the limb promordia shows that the limb ectoderm contains a level of ld transcripts fivefold higher relative to its mesenchyme. Furthermore, expression of ld transcripts in other parts of the developing embryo and in primitive streak embryos (gestational day 7) suggests possible roles for this gene in the earliest determinative events of morphogenesis. These data lead us to conclude that ld gene products are required for both proper AER differentiation and anteroposterior pattern formation in limb mesenchyme.
Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements.
Authors: Hanly S M , Rimsky L T , Malim M H , Kim J H , Hauber J , Duc Dodon M , Le S Y , Maizel J V , Cullen B R , Greene W C
Published: June 2007

The Rex proteins of types I and II human T-cell leukemia viruses (HTLV-I, HTLV-II) are required for expression of the viral structural gene products, gag and env and, thus, are essential for the replication of these pathogenic retroviruses. The action of Rex is sequence specific, requiring the presence of a cis-acting Rex response element located in the 3' long terminal repeat. This element corresponds to a predicted RNA secondary structure and functions in an orientation-dependent but position-independent manner. Rex acts through this response element to stimulate the nuclear export of the unspliced or singly spliced viral mRNA species encoding the virion structural proteins that are normally excluded from the cytoplasm. Although the Rex proteins of HTLV-I and HTLV-II can also function via the related Rev response element present in the env gene of the type I human immunodeficiency virus (HIV-1), the analogous HIV-1 Rev protein is unable to act on the HTLV-I Rex response element. This nonreciprocal pattern of genetic complementation by Rex and Rev suggests that these viral trans-regulators may interact directly with their RNA response elements.
Mammalian U2 snRNP has a sequence-specific RNA-binding activity.
Authors: Nelson K K , Green M R
Published: June 2007

The RNA branch formed during pre-mRNA splicing occurs at a wide variety of sequences (branch sites) in different mammalian pre-mRNAs. U2 small nuclear ribonucleoprotein (snRNP) binds to the pre-mRNA branch site following the interaction of a protein, U2AF, with the 3' splice site/polypyrimidine tract. Here we show that despite the variability of mammalian branch sites, U2 snRNP has a sequence-specific RNA-binding activity. Thus, RNA branch formation is regulated by two sequence-specific interactions: U2AF with the 3' splice site/polypyrimidine tract, and U2 snRNP with the branch site. The affinity of the branch site for U2 snRNP affects the efficiency of spliceosome assembly and splicing.
Activation of transcription by v-myb: evidence for two different mechanisms.
Authors: Klempnauer K H , Arnold H , Biedenkapp H
Published: June 2007

The retroviral oncogene v-myb encodes a nuclear, sequence-specific DNA-binding protein. To investigate the possibility that v-myb encodes a transcriptional regulator, we used a transient cotransfection assay to explore the potential of v-myb to influence the expression of other genes. We found that expression of a chicken lysozyme promoter/CAT gene construct was activated by v-myb in the presence of myb-specific binding sites. Action was not observed with a truncated v-myb protein lacking its DNA-binding domain. We also observed that expression of a hybrid human HSP70 promoter/CAT gene, lacking myb-specific binding sites, was activated by v-myb. However, in this case, the truncated v-myb protein, which lacked its DNA-binding domain, also activated HSP70/CAT expression, indicating that trans-activation of this gene construct was independent of the sequence-specific DNA-binding activity of the v-myb protein. These observations suggest that v-myb encodes a trans-activator and that activation of gene expression occurs by two different mechanisms, one of which involves specific binding of v-myb protein to the regulated gene.
Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.
Authors: Blanco J , Millstein L , Razik M A , Dilworth S , Cote C , Gottesfeld J
Published: June 2007

Immunoblotting experiments with polyclonal and monoclonal anti-transcription factor IIIA (TFIIIA) antibodies reveal different electrophoretic forms of TFIIIA in extracts from immature and mature oocytes of Xenopus laevis. The well-characterized 39-kD TFIIIA species is present in approximately 10(12) copies per cell in stage I-III previtellogenic oocytes and declines in abundance by 10- to 20-fold during oogenesis. An immunologically related protein of apparent molecular mass of 42 kD is present at 2-4% of the level of 39-kD TFIIIA in immature oocytes, and the level of this protein increases dramatically during oogenesis. Both the 39- and 42-kD proteins are complexed with 5S RNA in 7S ribonucleoprotein (RNP) particles. High-level transcription of the oocyte-type 5S genes in vitro requires 39-kD immature oocyte TFIIIA, whereas both 39-kD TFIIIA and the mature oocyte TFIIIA species of 42 kD support somatic-type 5S transcription. TFIIIA of 42 kD does not support oocyte-type 5S transcription in a fractionated transcription system derived from mature oocytes. Both proteins, however, bind the oocyte-type and somatic-type genes with comparable affinities and exhibit similar DNase footprints on both genes. These results suggest a model for the developmental regulation of 5S RNA gene transcription where 42-kD TFIIIA serves as an activator of somatic-type 5S transcription and as a repressor of oocyte-type transcription during early embryogenesis.
Specific expression of a novel cell wall hydroxyproline-rich glycoprotein gene in lateral root initiation.
Authors: Keller B , Lamb C J
Published: June 2007

A gene encoding a novel cell wall hydroxyproline-rich glycoprotein (HRGPnt3) was isolated from a genomic library of tobacco. The deduced protein (620 amino acids, Mr, 65,406) contains an amino-terminal hydrophobic signal peptide, is highly basic, and is rich in proline, although characteristic Ser-Pro4 repeats are found only beyond residue 204. At the carboxyl terminus, there is a 34-amino-acid unit repeated three times. Arginine is the predominant basic amino acid rather than lysine, as in other HRGPs. A second gene homologous to HRGPnt3 was revealed by Southern blot hybridization of tobacco genomic DNA. Northern blot hybridization identified a 1.9-kb transcript present at low levels in roots. To determine the underlying spatial pattern of expression, the HRGPnt3 promoter and the first 27 nucleotides of the open reading frame were fused to the beta-glucuronidase (GUS) reporter gene and transformed back into tobacco. Histochemical localization of GUS activity showed that the HRGPnt3 promoter was transiently induced in the pericycle and endodermis, specifically in the discrete, small subset of cells involved in the initiation of lateral roots. This pattern of expression, in cells destined to form the tip of the emerging lateral root, indicates that the encoded cell wall protein has a specialized structural function, possibly in the mechanical penetration of the cortex and epidermis of the main root, and that the HRGPnt3 promoter responds to an early morphogenetic signal for lateral root induction.
Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells.
Authors: Espeseth A S , Murphy S P , Linney E
Published: June 2007

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.
Cellular mosaicism in the methylation and expression of hemizygous loci in the mouse.
Authors: McGowan R , Campbell R , Peterson A , Sapienza C
Published: June 2007

Proposed models for the inheritance of locus-specific methylation phenotypes in somatic cells include those in which there is stable inheritance of a methylation pattern such that all cells contain a similarly methylated locus, as well as models in which the inheritance of methylation can be variable. We investigated these possibilities by examining the methylation and expression of hemizygous loci in the mouse. Our results demonstrate that differences in both methylation and expression can exist between apparently identical cells and that such mosaicism is genetically controlled.
Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements.
Authors: Dynlacht B D , Attardi L D , Admon A , Freeman M , Tjian R
Published: June 2007

In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells.