bluuebunny/crossref_metadata_2025 huggingface dataset:
Equipotent mouse ribosomal protein promoters have a similar architecture that includes internal sequence elements.
Authors: Hariharan N , Kelley D E , Perry R P
Published: June 2007

The promoters of the mouse ribosomal protein genes rpL30, rpL32, and rpS16 are of equal strength, as indicated by in vivo measurements of polymerase loading and by their relative efficiency in driving the expression of a linked reporter gene. The equipotency of these promoters appears to derive from a remarkably similar architecture in which five or more elements are distributed over a 200-bp region that spans a polypyrimidine-embedded cap site. Three trans-acting factors are shared by the rpL30 and rpL32 promoters, one of which, delta, recognizes a common CNGCCATCT motif in the first (untranslated) exons. Site-specific mutagenesis demonstrated that delta-factor binding is critical for rpL30 promoter function. The repeated occurrence of this novel promoter architecture among ribosomal protein genes with very different coding specificities is most readily explained by convergent evolution.
Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis.
Authors: Karmazyn-Campelli C , Bonamy C , Savelli B , Stragier P
Published: June 2007

During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.
The Hox-1.3 homeo box protein is a sequence-specific DNA-binding phosphoprotein.
Authors: Odenwald W F , Garbern J , Arnheiter H , Tournier-Lasserve E , Lazzarini R A
Published: June 2007

We report that the murine Hox-1.3 homeo domain protein is a nuclear phosphoprotein capable of binding to specific DNA sequences. DNase I protection of the Hox-1.3 gene promoter region with the Hox-1.3 protein identifies a binding site 144 bp upstream from the start of transcription. Both phosphorylated and nonphosphorylated forms bind DNA directly in a sequence-specific manner. Electrophoretic mobility shift assays were performed with a set of synthetic oligonucleotides representing either the DNase I-protected region of the Hox-1.3 gene or partially homologous sequences present in promoter regions of other characterized viral, yeast, and mammalian genes. From the results, we deduce a consensus binding motif of CPyPyNATTAT/GPy. Base substitutions in the core ATTA sequence severely reduce or abolish binding. In the SV40 enhancer, the Hox-1.3 binding motif overlaps both the octamer (Octa2) and the transactivator protein-1 (AP-1) binding sites. The Hox-1.3 binding motif also overlaps the nuclear factor III (NF-III) octamer motif in the adenovirus-2 origin of DNA replication. Overlap among DNA-binding sites suggests that regulation imparted by certain cis-elements may be integrated by these different factors.
xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.
Authors: Miller M , Kloc M , Reddy B , Eastman E , Dreyer C , Etkin L
Published: June 2007

The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system (CNS) of tadpoles and adult frogs. This cDNA (xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal (Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine (His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cell-specific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15-16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.
DNA bending by negative regulatory proteins: Gal and Lac repressors.
Authors: Zwieb C , Kim J , Adhya S
Published: June 2007

The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of gal and lac operator DNA sequences into a sensitive vector, pBend2. pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the gal operators, OE and OI. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously.
DNA-binding activity of the adenovirus-induced E4F transcription factor is regulated by phosphorylation.
Authors: Raychaudhuri P , Bagchi S , Nevins J R
Published: June 2007

Previous experiments have identified E4F, an inducible cellular factor that binds to sequences in the adenovirus E4 promoter that are critical for E1A-dependent transcriptional activation. The E4F factor has been purified and shown to stimulate transcription in vitro from the E4 promoter. Analysis of the affinity-purified factor identifies a single polypeptide of 50 kD that has E4F-specific binding activity. E4F binding activity is also regulated during F9 cell differentiation and can be activated in differentiated F9 cells by viral infection. Furthermore, the activation process appears to involve a phosphorylation event, because treatment of E4F with alkaline phosphatase abolishes activity and incubation of the phosphatase-inactivated factor with an extract from virus infected cells restores activity.
Positive and negative regulation of the gene for transcription factor IIIA in Xenopus laevis oocytes.
Authors: Scotto K W , Kaulen H , Roeder R G
Published: June 2007

Expression of the positively acting 5S gene-specific transcription factor, TFIIIA, is regulated during development, with highest levels of mRNA and protein occurring during oogenesis. By analysis of TFIIIA promoter mutants microinjected into late stage Xenopus oocytes, we have determined DNA sequences required for the transcription of this gene and we have identified proteins that bind to these regulatory sequences. A negative element lies between positions -306 and -289. Three positive-acting sequences are located between positions -289 and -253, -250 and -173, and -144 and -101. Gel shift analyses of TFIIIA promoter fragments incubated with Xenopus oocyte extracts have identified two DNA-protein complexes. One complex, designated B1, requires sequences within the promoter region extending from -271 to -253 while the second complex, designated B2, involves promoter sequences from -235 to -221. The protein involved in formation of the B1 complex has been found to be related to the human adenovirus major late transcription factor, USF.
Developmental and tissue-specific regulation of a novel transcription factor of the sea urchin.
Authors: Barberis A , Superti-Furga G , Vitelli L , Kemler I , Busslinger M
Published: June 2007

We have identified a novel transcription factor that interacts with the promoter of four tissue-specific late histone H2A-2 and H2B-2 genes of the sea urchin by DNase I footprint, mobility shift, and methylation interference analyses. The binding site for this factor is required for efficient transcription of the H2B-2.1 gene both in vitro in nuclear extracts of gastrula embryos and in vivo in microinjected sea urchin embryos. This factor binds with equal affinity to the recognition sequences of all four histone genes in cross-competition assays. Moreover, the binding site of the H2B-2.2 promoter can functionally substitute for that of the H2B-2.1 gene in in vivo expression experiments. Nevertheless, all four binding sites share little sequence homology with each other. This transcription factor increases in abundance during embryogenesis and has been detected in the adult sea urchin only in the tube feet, where the late H2A-2 and H2B-2 genes are expressed specifically. Therefore, we refer to this factor as tissue-specific activator protein (TSAP). The close correlation between the presence of TSAP and the expression pattern of the late H2A-2 and H2B-2 genes suggests that this transcription factor is directly responsible for the developmental and tissue-specific regulation of these genes.
Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice.
Authors: Swift G H , Kruse F , MacDonald R J , Hammer R E
Published: June 2007

The 134-bp enhancer region of the pancreatic elastase I gene is sufficient to direct pancreatic acinar cell-specific transcription in transgenic mice and in transfected cells in culture. Ten-base-pair scanner mutations in three separate enhancer domains that inactivate enhancer function in transfected pancreatic cells in culture have no significant effect in transgenic mice. Because any pair of the three domains is sufficient to direct pancreas-specific expression in mice, no one domain is required for pancreas-specific transcription. Disruption of any two domains does inactivate the enhancer function in transgenic mice. Therefore, the elastase I enhancer domains essential for function in transfected cells in culture are not essential in animals but have a redundant function not apparent in transfected cells. This redundant function is not because of the particular acinar cell line used for transfections, the nature of the reporter gene, or the state of integration of the foreign test gene. We conclude that a trans-acting transcription factor or a modification of a factor(s) present in pancreatic cells of an animal is absent in pancreatic acinar cell lines.
Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development.
Authors: Brenner C A , Adler R R , Rappolee D A , Pedersen R A , Werb Z
Published: June 2007

Extracellular matrix (ECM) remodeling accompanies cell migration, cell-cell interactions, embryo expansion, uterine implantation, and tissue invasion during mammalian embryogenesis. We have found that mouse embryos secrete functional ECM-degrading metalloproteinases, including collagenase and stromelysin, that are inhibitable by the tissue inhibitor of metalloproteinases (TIMP) and that are regulated during peri-implantation development and endoderm differentiation. mRNA transcripts for collagenase, stromelysin, and TIMP were detected as maternal transcripts in the unfertilized egg, were present at the zygote and cleavage stages, and increased at the blastocyst stage and with endoderm differentiation. These data suggest that metalloproteinases function in cell-ECM interactions during growth, development, and implantation of mammalian embryos.
A 3' exonuclease activity degrades the pseudogene 5S RNA transcript and processes the major oocyte 5S RNA transcript in Xenopus oocytes.
Authors: Xing Y Y , Worcel A
Published: June 2007

Transcription of the major oocyte 5S RNA gene (o) and pseudogene (psi) of Xenopus laevis yields different RNAs with three different homologous systems: oocyte microinjection, whole oocyte extract, and fractionated TFIIIA + TFIIIB + TFIIIC components. Those peculiar results are caused by a 3' RNA exonuclease activity, which is inhibited in the oocyte extract, that rapidly degrades the pseudogene 5S RNA but does not degrade as readily the chimeric RNA transcripts generated by HindIII-truncated 5S RNA pseudogenes. The same, or a similar, RNase activity processes the 130- and the 142-base-long transcripts of the major oocyte 5S RNA gene into mature 120-base-long 5S RNA. We performed site-specific mutagenesis on the somatic 5S RNA gene and changed specific nucleotides on the somatic 5S RNA. These studies indicated that the structure that confers stability to the 5S RNA in vivo and in vitro is the 9-bp helix formed in 5S RNA, but not in psi 5S RNA, by the complementary 5' and 3' ends of the molecule.
The secE gene encodes an integral membrane protein required for protein export in Escherichia coli.
Authors: Schatz P J , Riggs P D , Jacq A , Fath M J , Beckwith J
Published: June 2007

Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli. These genes are secA, secD, prlA/secY, and secE. We discuss the significance of the failure to find new sec genes after extensive use of this approach. One of the genes, secE, has been characterized in some detail. From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein. The data presented here and in the accompanying paper strongly suggest that secE has an important role in E. coli protein export.
Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.
Authors: Birkenmeier E H , Gwynn B , Howard S , Jerry J , Gordon J I , Landschulz W H , McKnight S L
Published: June 2007

This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.
Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer.
Authors: Forsburg S L , Guarente L
Published: June 2007

The CYC1 gene of Saccharomyces cerevisiae is positively regulated by the HAP2 and HAP3 proteins, which form a heteromeric complex that binds to a CCAAT box in the upstream activation site, UAS2, and which activate transcription in a nonfermentable carbon source. We carried out a genetic analysis to identify additional trans-acting regulatory factors exerting their effects through UAS2. We present the identification and characterization of a new locus, HAP4, which is shown to encode a subunit of the DNA-binding complex at UAS2. In the hap4 mutant, the binding of HAP2 and HAP3 (HAP2/3) is not observed in vitro. The HAP4 gene is regulated transcriptionally by a carbon source, suggesting that it encodes a regulatory subunit of the bound complex. The sequence of HAP4 shows a highly acidic region, which innactivated the protein when deleted. Replacement of this region with the activation domain of GAL4 restored activity, suggesting that it provides the principal activation domain to the bound HAP2/3/4 complex.
Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control.
Authors: Miller P F , Hinnebusch A G
Published: June 2007

Translational control of the GCN4 gene of Saccharomyces cerevisiae requires at least two of the four short upstream open reading frames (URFs) in the leader of GCN4 mRNA. URF4 is a strong negative element that is sufficient for repression of GCN4 expression in normal growth conditions. URF1 is approximately 30-fold less effective as a translational barrier when it is the single URF present in the mRNA leader and is required upstream from URF4 for efficient derepression of GCN4 expression under amino acid starvation conditions. We show that the last codon plus 10 bp immediately after the stop codon of URF4 are sufficient to convert URF1 into a strong translational barrier when it is present as a solitary URF. This result suggests that the characteristics of translation termination at URF4 are responsible largely for its strong inhibitory effect on translation initiation at the GCN4 AUG codon. Introduction of the same URF4 sequences at URF1 also reduces GCN4 expression under derepressing conditions when URF1 is upstream from URF4. This fact suggests that URF1 translation normally is compatible with efficient scanning and initiation downstream and that this property is required for its ability to overcome the translational barrier at URF4. These findings are consistent with the idea that ribosomes must first translate URF1 and then resume scanning in order to traverse URF4 sequences under starvation conditions. Our results indicate that nucleotides located 3' to the stop codon can be as important as those located 5' to the start site in determining the inhibitory effect of an URF on translation initiation downstream.
Antigenic diversity by the recombination of pseudogenes.
Authors: Thon G , Baltz T , Eisen H
Published: June 2007

During the course of an infection, the hemoflagellate Trypanosoma equiperdum sequentially expresses an extensive repertoire of surface glycoproteins. There is evidence that combinations of silent genes are involved in the generation of this repertoire, but the combination rules are not known. To gain insight into these rules, we determined the fine structure of a composite gene. The gene coding for the variant surface glycoprotein 20 of T. equiperdum is a late gene generated by the partial duplication of three silent pseudogenes. Two closely related but not identical '5' donors' form a mosaic coding for the antigenic portion of the protein. A telomeric '3' donor' provides the last 200 nucleotides of the expressed gene. The sequences of the 5' and 3' donors are not related except for a short segment in which the hybrid junction is formed. These results demonstrate that recombinational processes generate diversity by reassorting sequences and also allow the expression of pseudogenes. Furthermore, the use of a short sequence similarity for the formation of the 5'-3' donor hybrid suggests a mechanism that may act in ordering the expression of the variant surface glycoproteins.
Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.
Authors: Bier E , Vaessin H , Shepherd S , Lee K , McCall K , Barbel S , Ackerman L , Carretto R , Uemura T , Grell E
Published: June 2007

A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.
cappuccino and spire: two unique maternal-effect loci required for both the anteroposterior and dorsoventral patterns of the Drosophila embryo.
Authors: Manseau L J , Schüpbach T
Published: June 2007

cappuccino and spire are unique Drosophila maternal-effect loci that participate in pattern formation in both the anteroposterior and dorsoventral axes of the early embryo. Mutant females produce embryos lacking pole cells, polar granules, and normal abdominal segmentation. They share these defects with the posterior group of maternal-effect genes. Although embryos are defective in abdominal segmentation, in double mutant combinations with Bicaudal D, abdominal segments can be formed in the anterior half of the egg. This indicates that embryos produced by mutant females contain the 'posterior determinant' required for abdominal segmentation (Nüsslein-Volhard et al. 1987) and suggests that the wild-type gene products are not required for production of the posterior determinant but, rather, for its localization or stabilization. The vasa protein, a component of polar granules, is not localized at the posterior pole of mutant egg chambers or embryos, providing additional support for the hypothesis that localization to or stabilization of substances at the posterior pole of the egg chamber is defective in mutant females. Females mutant for the strongest alleles also produce dorsalized embryos. Phenotypic analysis reveals that these dorsalized embryos also have abdominal segmentation defects. The mutant phenotypes can be ordered in a series of increasing severity. Pole cell formation is most sensitive to loss of functional gene products, followed by abdominal segmentation, whereas normal dorsoventral patterning is the least sensitive to loss of functional gene products. In addition, mutant females contain egg chambers that appear to be dorsalized, resulting in the production of eggs with dorsalized eggshells. Germ-line mosaics indicate that cappuccino and spire are required in the oocyte-nurse cell complex. This suggests that the eggshell phenotype results from altered pattern in the underlying germ cell. Also, we defined the epistatic relationships between several early patterning loci, on the basis of an analysis of the eggs and embryos produced by females doubly mutant for cappuccino or spire and other loci that affect the pattern of both the egg and the embryo. On the basis of our current knowledge of the genes involved in this process, we formulated a working model for the early steps in dorsoventral patterning.
Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.
Authors: Erickson J W , Gross C A
Published: June 2007

The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.
Half helical turn spacing changes convert a frog into a mouse rDNA promoter: a distant upstream domain determines the helix face of the initiation site.
Authors: Pape L K , Windle J J , Sollner-Webb B
Published: June 2007

Transcription of frog rDNA by mouse cell factors is the only documented exception to the observed species selectivity of rRNA gene expression. This heterologous transcription is authentic in that it uses the normal frog upstream and core promoter domains, as well as the normal mouse polymerase I transcription factors, but it initiates at residue -4. We now show that by introducing an insertion or deletion of approximately one-half helical turn anywhere within the 90-bp region between the upstream and core promoter domains, the initiation site moves to residue +1. Promoters bearing spacing changes of approximately one or two full helix turns do not initiate at residue +1, whereas a promoter with a one and one-half-turn helical turn spacing change again supports initiation at residue +1. Thus, the position of the upstream domain of the frog promoter shows a stereo-specific requirement relative to the core promoter domain and dictates the face of the DNA helix on which transcription initiates, 140 bp away. In contrast, relative to the core promoter domain, initiation can occur on either side of the DNA helix. Furthermore, the striking observation that several frog half helical turn spacing change mutants are stronger templates with the mouse factors than the homologous mouse rDNA suggests that the polymerase I transcriptional machinery of even distantly related species is far more similar than generally envisioned.
Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA.
Authors: Helfman D M , Roscigno R F , Mulligan G J , Finn L A , Weber K S
Published: June 2007

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.
Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae.
Authors: Briza P , Breitenbach M , Ellinger A , Segall J
Published: June 2007

During sporulation of Saccharomyces cerevisiae, the four haploid nuclei generated by meiosis are encapsulated within multilayered spore walls. Taking advantage of the natural fluorescence imparted to yeast spores by the presence of a dityrosine-containing macromolecule in the spore wall, we identified and cloned two genes, termed DIT1 and DIT2, which are required for spore wall maturation. Mutation of these genes has no effect on the efficiency of spore formation or spore viability. The mutant spores, however, fail to accumulate the spore wall-specific dityrosine and lack the outermost layer of the spore wall. The absence of this cross-linked surface layer reduces the resistance of the spores to lytic enzymes, to ether, and to elevated temperature. Expression of the DIT and DIT2 genes is restricted to sporulating cells, with the DIT1 transcripts accumulating at the time of prospore enclosure and just prior to the time of dityrosine biosynthesis. Both genes act in a spore-autonomous manner implying that at least some of the activities responsible for forming the outermost layer of the spore wall reside within the developing spore rather than in the surrounding ascal cytoplasm. As the DIT2 gene product has significant homology with cytochrome P-450s, DIT2 may be responsible for catalyzing the oxidation of tyrosine residues in the formation of dityrosine.
Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1.
Authors: Martin D I , Orkin S H
Published: June 2007

The murine, erythroid DNA-binding protein GF-1 (also known as NF-E1, Eryf 1), a 413-amino acid polypeptide with two novel finger domains of the Cx-Cx variety, recognizes a consensus GATA motif present in cis elements of the majority of erythroid-expressed genes. We have performed a structure-function analysis of this protein to evaluate its potential as a transcriptional activator and to examine the role of the finger domains in DNA binding. Using a cotransfection assay, we find that GF-1 is a potent transcriptional activator with several activation domains but that this is revealed only in heterologous cells and with reporters containing minimal promoters onto which either a single or multiple GATA-binding sites are placed. The two fingers of GF-1 are functionally distinct and cooperate to achieve specific, stable DNA binding. The amino finger is necessary only for full specificity and stability of binding, whereas the carboxyl finger is required for binding. The role of each finger is more pronounced with some GATA-binding sites than with others, suggesting a diversity of interactions between GF-1 and different target sites. The complex activation and DNA-binding properties of GF-1 are likely to contribute to the ability of this single protein to participate widely in gene expression throughout erythroid development.
A plant DNA-binding protein increases the number of active preinitiation complexes in a human in vitro transcription system.
Authors: Katagiri F , Yamazaki K , Horikoshi M , Roeder R G , Chua N H
Published: June 2007

TGA1a is a tobacco DNA-binding protein that binds to the activation sequence-1 (as-1) element of the cauliflower mosaic virus 35S promoter. We have produced TGA1a in Escherichia coli, purified it from bacterial extracts, and examined its effect on transcription in a human in vitro system. Addition of TGA1a stimulates transcription by up to 20 times, and the stimulation is dependent on the presence of the as-1 element in the promoter. When transcription reinitiation is inhibited by 0.3 M KCl, activation is similar. Therefore, TGA1a activates transcription by increasing the number of active preinitiation complexes. After formation of the preinitiation complexes in the presence of TGA1a, oligonucleotides containing TGA1a-binding sites do not significantly affect the stimulated level of transcription. This result indicates that a complex remains committed to the promoter site after initiation and that this complex is used repeatedly during several initiation events. Our study demonstrates for the first time that a plant factor can activate transcription in a human in vitro system and that the activation mechanism of the plant factor is similar to that of mammalian factors.
Endonucleolytic cleavage of a maternal homeo box mRNA in Xenopus oocytes.
Authors: Brown B D , Harland R M
Published: June 2007

We have identified a messenger RNA (mRNA) sequence from a Xenopus homeo box-containing gene that is the target for a sequence-specific endoribonuclease in vivo. Synthetic RNA transcribed from an allele of the maternal gene Xlhbox2B is efficiently cleaved when injected into Xenopus oocytes. The cleavage sequence lies between the protein-coding region and a 600-base 3'-untranslated region. Intermediates in degradation are readily observed: Both the 5' and 3' products of cleavage are recovered, thus showing that the cleavage activity is an endonuclease. When a 90-base region of the Xlhbox2B sequence is inserted into a second homeo box RNA that is normally stable, it is sufficient to confer an identical cleavage reaction on the hybrid RNA. The cleaved region contains a repeated sequence motif and is cut at multiple sites. Inhibition of translation does not affect the rate or extent of cleavage, while the coinjection of antisense RNA complementary to the 90-base region completely blocks the reaction. Because most mRNAs are not found on polysomes during oogenesis, translation-independent cleavage at such sites may provide a novel post-transcriptional mechanism to regulate the amount of mRNA available for embryogenesis.
Pairing of homologous DNA sequences by proteins: evidence for three-stranded DNA.
Authors: Hsieh P , Camerini-Otero C S , Camerini-Otero R D
Published: June 2007

We show that recombinases form joint molecules over very short regions of homology. When these molecules are deproteinized the three strands are in a structure that is surprisingly resistant to dissociation by branch migration, even at elevated temperatures. The joint molecules dissociate at temperatures comparable to those required to melt DNA duplexes of the same length and sequence. We also show that nonenzymatically formed structures of the same length and sequence, which have a free third strand ready to branch migrate, dissociate at much lower temperatures. These results provide compelling evidence that the three DNA strands in the region of pairing are hydrogen bonded to each other. Our observations suggest that such a novel three-stranded DNA molecule, or a structure very similar to it, may be the intermediate in general recombination that is used in the recognition of sequence homology. We discuss some of the structural features implicit in this molecule containing any base sequence and compare them with those manifest in true DNA triple helices containing special sequence motifs.
The 65-kD subunit of NF-kappa B is a receptor for I kappa B and a modulator of DNA-binding specificity.
Authors: Urban M B , Baeuerle P A
Published: June 2007

A recent study has shown that the dimeric form of the 50-kD DNA-binding subunit of the NF-kappa B transcription factor is commonly associated with two molecules of a 65-kD protein (p65) and that p65 appears to be required for inactivation of the p50 dimer by the inhibitory subunit I kappa B. Here, we provide evidence that p65 serves as a receptor for I kappa B. Preincubation of I kappa B with purified p65 prevented I kappa B from inactivating the heterotetrameric form of NF-kappa B. Furthermore, excess p65 could very efficiently activate the latent form of NF-kappa B composed of p50, p65, and I kappa B, presumably by binding the I kappa B, which was released from the complex due to its inherent off rate. An additional function of p65 in modulating the DNA-binding specificity of p50 was found. In the absence of p65, p50 could bind with high affinity to completely palindromic sites. The heterotetramer recognized these sites with an affinity greater than 10-fold lower but preferred the less symmetric physiological kappa B site. The affinity of p50 for the most frequent kappa B motif 5'-GGGACTTTCC-3' was enhanced twofold by p65 to yield a dissociation constant of approximately 4 x 10(-13) M. This study describes novel functions for a non-DNA-binding accessory protein of a transcription factor in controlling its inducibility and DNA-binding specificity.
Regulation of a human epidermal keratin gene: sequences and nuclear factors involved in keratinocyte-specific transcription.
Authors: Leask A , Rosenberg M , Vassar R , Fuchs E
Published: June 2007

The keratinocyte is a major cell type of the body, and in epidermis, keratinocytes have potential for future gene targeting and drug therapy. Despite the importance of keratinocytes in cell biology and medicine, little is known about the molecular mechanisms underlying keratinocyte-specific gene expression. Here, we report the first detailed characterization of the sequences and factors controlling expression of a human gene expressed specifically in keratinocytes. Using 5' upstream sequence of the human K14 keratin gene coupled to one of two reporter genes, we examined sequences necessary and sufficient for expression of K14 in both cultured human keratinocytes and in mitotically active basal keratinocytes of transgenic mouse epidermis. We demonstrated the existence of distal and proximal elements located 5' from the transcription initiation site of the hK14 gene, which when combined with a TATA box element, appear to act in concert to drive keratinocyte-specific expression. We examined the proximal region in detail. After using CAT assays to narrow a transcriptional activation element to within 110 bp, we demonstrated the existence of a keratinocyte nuclear factor which binds to a 10-bp palindrome, 5'-GCCTGCAGGC-3', within this domain. Using methylation interference analysis, we identified the G residues important for factor binding, and showed that point mutations in these G residues not only blocked factor binding but also resulted in decreased transcriptional activity of an hK14-CAT gene. The factor was most abundant in keratinocytes, was expressed at lower levels in some simple epithelial cell lines, and was not detected in fibroblasts or lymphoma cells. Moreover, the 10-bp sequence was similar to sequences found in the 5' upstream sequences of several other genes expressed in keratinocytes, and at least one of these genes, the human K1 gene, contained a sequence that competed with the hK14 proximal element for binding factor. Collectively, our data suggest that both the sequence and the nuclear factor that we have identified may be involved in controlling keratinocyte-specific expression in vitro and in vivo.
Gene targeting at the human CD4 locus by epitope addition.
Authors: Jasin M , Elledge S J , Davis R W , Berg P
Published: June 2007

Homologous recombination at the CD4 locus in a human T-cell line has been achieved by an approach called epitope addition. The endogenous CD4 gene provided transcription, translation, and leader sequences to a crippled introduced Thy-1 gene, resulting in the expression of murine Thy-1 epitopes on the surface of the human cells. Thy-1+ cells were selected using the Fluorescence Activated Cell Sorter (FACS). An estimated 700-fold enrichment for homologous versus nonhomologous integration events was obtained, such that 70% of cells scoring positive for Thy-1 were derived from gene targeting. Three of the Thy-1+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.
Clonal and systemic analysis of long-term hematopoiesis in the mouse.
Authors: Jordan C T , Lemischka I R
Published: June 2007

We have analyzed the temporal in vivo fate of 142 individual stem cell clones in 63 reconstituted mice. Long-term sequential analyses of the four major peripheral blood lineages, obtained from animals engrafted with genetically marked stem cells, indicate that developmental behavior is primarily a function of time. As such, the first 4-6 months post-engraftment is characterized by frequent fluctuations in stem cell proliferation and differentiation behavior. Gradually, a stable hematopoietic system emerges, dominated by a small number of totipotent clones. We demonstrate that single stem cell clones are sufficient to maintain hematopoiesis over the lifetime of an animal and suggest that mono- or oligoclonality may be a hallmark of long-term reconstituted systems. A model is proposed, wherein lineage-restricted differentiation and dramatic clonal flux are consequences of mechanisms acting on an expanding pool of totipotent cells and are not indicative of intrinsically distinct stem cell classes.
Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast.
Authors: Ammerer G
Published: June 2007

In yeast the alpha-specific regulators, alpha 1 and alpha 2 have been proposed to be DNA-binding proteins, both of which have to interact with an additional factor called PRTF or GRM, respectively, to exert their biological functions. Although the cis-acting sequence requirements for alpha 1 and alpha 2 are different, their target sequences share a common motif. PRTF or GRM is thought to act via this common DNA sequence; therefore, it has been suggested that they represent the same factor. I purified a protein that binds to this common promoter element by DNA affinity chromatography. The purified protein is able to recruit the alpha-specific activator alpha 1 to its binding sites, suggesting that it is indeed PRTF. Further evidence is presented to show that PRTF and GRM are the same protein and that PRTF plays a role in the activation of a-specific genes. Specific antibodies to the purified protein were obtained. They identify the protein as a component of DNA-protein complexes that formed with cell-type-specific promoter sequences. Using these antibodies, the gene encoding the protein was cloned from a yeast lambda gt11 expression library. The DNA sequence established that the gene encoding PRTF/GRM is identical with a previously described gene, FUN80 (essential factor of unknown function) or MCM1 (minichromosome maintenance). Sequence comparison showed further that PRTF/GRM shares similarities with a repressor from yeast, ARGRI, and the mammalian transcription factor SRF.
Human gamma- to beta-globin gene switching in transgenic mice.
Authors: Behringer R R , Ryan T M , Palmiter R D , Brinster R L , Townes T M
Published: June 2007

Previous studies demonstrated correct tissue- and temporal-specific expression of human gamma- and beta-globin genes in transgenic mice; however, expression was extremely low. When the erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located upstream of the human beta-globin locus were fused individually to gamma- or beta-globin genes, expression increased to endogenous mouse globin levels but temporal specificity was lost. In contrast, when the HS sequences were combined with fragments containing both gamma- and beta-globin genes, correct developmental regulation was restored. We suggest that human gamma- to beta-globin gene switching during development results from competition of individual globin gene family members for interaction with the HS sequences and that factors influencing these competitive interactions determine temporal specificity.
A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator.
Authors: Chasman D I , Lue N F , Buchman A R , LaPointe J W , Lorch Y , Kornberg R D
Published: June 2007

GRF2, an abundant yeast protein of Mr approximately 127,000, binds to the GAL upstream activating sequence (UASG) and creates a nucleosome-free region of approximately 230 bp. Purified GRF2 binds to sequences found in many other UASs, in the 35S rRNA enhancer, at centromeres, and at telomeres. Although GRF2 stimulates transcription only slightly on its own, it combines with a neighboring weak activator to give as much as a 170-fold enhancement. This effect of GRF2 is strongly distance-dependent, declining by 85% when 22 bp is interposed between the GRF2 and neighboring activator sites.
A wide variety of DNA sequences can functionally replace a yeast TATA element for transcriptional activation.
Authors: Singer V L , Wobbe C R , Struhl K
Published: June 2007

We created a library of DNA molecules in which the required TATA element of a yeast gal-his3 promoter is replaced by random-sequence oligomers averaging 16 bp in length. Surprisingly, 1% of such random sequences functionally replace the native yeast TATA element. In many cases, sequences completely unrelated to the consensus TATA element (TATAAA) stimulate transcription with equal or increased efficiency. Transcription mediated by these synthetic elements requires GAL4 and is initiated from normal his3 initiation sites, suggesting that it occurs by a mechanism indistinguishable from that involving wild-type TATA elements. Many, but not all, of these elements act as substrates for yeast TFIID in reconstituted transcription reactions in vitro. These observations indicate that yeast TFIID can stimulate transcription from TATA elements whose sequences differ from the consensus, and they suggest the possibility of alternative factors that may provide a related function for transcriptional activation.