bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteins that bind to Drosophila chorion cis-regulatory elements: a new C2H2 zinc finger protein and a C2C2 steroid receptor-like component.
Authors: Shea M J , King D L , Conboy M J , Mariani B D , Kafatos F C
Published: June 2007

Gel mobility-shift assays have been used to identify proteins that bind specifically to the promoter region of the Drosophila s15 chorion gene. These proteins are present in nuclear extracts of ovarian follicles, the tissue where s15 is expressed during development, and bind to specific elements of the promoter that have been shown by transformation analysis to be important for in vivo expression. The DNA binding specificity has been used for molecular cloning of two components from expression cDNA libraries and for their tentative identification with specific DNA-binding proteins of the nuclear extracts. The mRNAs for both of these components, CF1 and CF2, are differentially enriched in the follicles. DNA sequence analysis suggests that both CF1 and CF2 are novel Drosophila transcription factors. CF2 is a member of the C2H2 family of zinc finger proteins, whereas CF1 is a member of the family of steroid hormone receptors. The putative DNA-binding domain of CF1 is highly similar to the corresponding domains of certain vertebrate hormone receptors and recognizes a region of DNA with similar, hyphenated palindromic sequences. The nature of CF1 raises the possibility of hormonal control of choriogenesis in Drosophila.
Cloning of the Schizosaccharomyces pombe TFIID gene reveals a strong conservation of functional domains present in Saccharomyces cerevisiae TFIID.
Authors: Hoffmann A , Horikoshi M , Wang C K , Schroeder S , Weil P A , Roeder R G
Published: June 2007

The gene encoding the Schizosaccharomyces pombe TATA box-binding factor (TFIID) was cloned and sequenced. The gene contains three introns and codes for a polypeptide of 231 amino acids. The cDNA-expressed protein showed both TATA box-binding and basal transcription activities. The carboxy-terminal three-quarters of S. pombe TFIID shares an extraordinary degree of amino acid sequence homology with a corresponding region of Saccharomyces cerevisiae TFIID that has been shown to be necessary and sufficient for TATA box-binding and basal transcription activities. In contrast, the amino-terminal regions of the S. pombe and S. cerevisiae TFIIDs differ markedly in amino acid sequence and composition. Structure and function relationships of TFIID are discussed in light of these data.
Adenovirus terminal protein mediates both nuclear matrix association and efficient transcription of adenovirus DNA.
Authors: Schaack J , Ho W Y , Freimuth P , Shenk T
Published: June 2007

Adenovirus DNA is tightly bound to the nuclear matrix throughout the course of infection. Analysis of adenovirus DNA from infected HeLa cell nuclei after extraction with lithium diiodosalicylate and digestion with restriction enzymes demonstrated that the sites of tightest attachment occur in the terminal fragments of the linear viral chromosome. Analysis of viruses mutated in the precursor terminal protein coding sequence demonstrated that the terminal protein, which is covalently attached to the 5' end of each DNA strand, mediates the tight binding. Virions containing chromosomes with mutant terminal proteins were unpackaged and viral DNA accumulated in the nucleus at a normal rate and competed for the limiting component during transcription complex formation, but their early genes were transcribed at reduced efficiency by both RNA polymerases II and III. The transcriptional defects were not complemented by coinfection with a wild-type virus. We propose that the adenovirus chromosome may exist as a single chromatin domain during infection and that binding of DNA to the nuclear matrix may play a critical role in adenovirus transcription.
Induction by HMBA and DMSO of genes introduced into mouse erythroleukemia and other cell lines by transient transfection.
Authors: Campbell P L , Kulozik A E , Woodham J P , Jones R W
Published: June 2007

We have found rapid induction of various genes, including human globin genes, in response to hexamethylene bisacetamide (HMBA) and dimethyl sulfoxide (DMSO) in transiently transfected cells. In mouse erythroleukemia cells (MELCs), this effect is detected within 1 hr of exposure of the cells to inducer before the endogenous mouse globin genes are induced. It does not require protein synthesis and is reversed if the inducer is removed. This and other evidence suggest that the mechanism involves a change in activity of a factor intimately involved with transcription, probably as a result of post-translational modification. As such, it may represent an early triggering event in terminal differentiation, and its relevance to the expression of human globin genes in stable transfectants and to induction of the mouse globin genes is discussed. Other cell lines (K562 and NSO) also show this response, which may therefore involve a ubiquitous mechanism. We also found that HMBA depresses the expression of endogenous globin genes in K562, the opposite of this differentiation inducer's effect on MELC.
In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1.
Authors: Pfeifer G P , Tanguay R L , Steigerwald S D , Riggs A D
Published: June 2007

The promoter region of the X-linked human phosphoglycerate kinase-1 (PGK-1) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomic sequencing study in which 450 bp of the human PGK-1 promoter region was analyzed for the presence of in vivo protein footprints and cytosine methylation at all CpG sites. A technique was devised to selectively visualize the DNA of the inactive X chromosome (Xi), even in the presence of the active X chromosome (Xa). We found that the human Xa in both normal male lymphocytes and hamster-human hybrids is completely unmethylated at all 120 CpG sites. In contrast, 118 of the CpG sites are methylated on the human Xi in hamster-human hybrids. The Xi in normal female lymphocytes is also highly methylated, but some GCG or CGC trinucleotides partially escape methylation; all other CpGs are fully methylated. In vivo footprinting studies with dimethylsulfate (DMS) revealed eight regions of apparent protein-DNA contacts on the Xa. Four of the footprints contained the consensus sequence of the binding site for transcription factor Sp1. The other regions include potential binding sites for transcription factors ATF, NF1, and a CCAAT-binding protein. The Xi did not show any specifically protected sequences, and with the exception of four hyperreactive sites, the in vivo DMS reactivity profile of Xi DNA was very similar to that of purified, linear Xi DNA. The implications of these findings with regard to the maintenance of methylation-free islands, X chromosome inactivation, and the chromatin structure of facultative heterochromatin are discussed.
Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression.
Authors: Carding S R , Kyes S , Jenkinson E J , Kingston R , Bottomly K , Owen J J , Hayday A C
Published: June 2007

The gamma delta T-cell receptor (TCR) is the first TCR to be expressed in ontogeny in all vertebrates in which it has been examined thoroughly. Murine gamma delta cell-surface protein is detected by the fourteenth day of gestation. In this work, the activation of gamma delta RNA has been studied. Data indicate that the first TCR protein to appear in the thymus is encoded by gamma genes that are activated after cells colonize the thymus. However, the sequential appearance of different gamma delta TCR proteins during thymic ontogeny cannot be readily explained by differential temporal activation of V gamma genes in the thymus. There are distinct patterns of gamma and delta gene expression during fetal liver development and in the fetal gut (or tissue associated with it). Cells apparent in the liver of mice at birth express gamma delta cell-surface protein, but they disappear from the liver very soon afterward. One V gamma gene is rearranged and expressed prethymically. In addition, gamma gene expression is detectable in the livers of newborn athymic mice. Together, these observations indicate a thymic-independent pathway of activation of TCR genes.
Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer.
Authors: Keller S A , Rosenberg M P , Johnson T M , Howard G , Meisler M H
Published: June 2007

We localized the cis-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to approximately 2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTF1. The close association of the PTF1-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTF1 in diabetic pancreas. PTF1-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.
Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Authors: Richardson H E , Stueland C S , Thomas J , Russell P , Reed S I
Published: June 2007

The Cks1 protein is a component of the Cdc28 protein kinase in the budding yeast Saccharomyces cerevisiae. This paper reports the cloning of two homologs of the S. cerevisiae CKS1 gene from human cells. These homologs, CKShs1 and CKShs2, both encode proteins of 79 amino acids that share considerable homology at the amino acid level with the products of CKS1 from S. cerevisiae and suc1+ from the fission yeast Schizosaccharomyces pombe. Both human homologs are capable of rescuing a null mutation of the S. cerevisiae CKS1 gene when expressed from the S. cerevisiae GAL1 promoter. S. pombe suc1+ expressed from the GAL1 promoter is also capable of rescuing a S. cerevisiae cks1 null mutation. Ckshs1 or Ckshs2 protein linked to Sepharose beads can bind the Cdc28/Cdc2 protein kinase from both S. cerevisiae and human cells. The CKShs1 and CKShs2 mRNAs are expressed in different patterns through the cell cycle in HeLa cells, which may reflect specialized roles for the encoded proteins.
Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position.
Authors: Brow D A , Guthrie C
Published: June 2007

Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.
The b mating-type locus of Ustilago maydis contains variable and constant regions.
Authors: Kronstad J W , Leong S A
Published: June 2007

The b locus of the phytopathogenic fungus Ustilago maydis encodes a multiallelic recognition function that controls the ability of the fungus to form a dikaryon and complete the sexual stage of the life cycle. The b locus has at least 25 alleles and any combination of two different alleles, brought together by mating between haploid cells, allows the fungus to cause disease and undergo sexual development within the plant. An open reading frame of 410 amino acids has been shown to specify a polypeptide responsible for the activity of the b1 allele, and comparisons of the predicted amino acid sequences for 6 b alleles allowed identification of variable and constant regions within the coding region of the gene. Haploid strains carrying a null mutation at the b locus, created by gene disruption, are viable but fail to interact with formerly compatible strains to give an infectious dikaryon. Analysis of mutants carrying a null allele indicated that the products of different alleles of the b locus combine to form a new regulatory activity and that this activity directly or indirectly turns on the pathway leading to sexual development and pathogenesis.
Identification of two polypeptide segments of CCAAT/enhancer-binding protein required for transcriptional activation of the serum albumin gene.
Authors: Friedman A D , McKnight S L
Published: June 2007

We used molecular genetic methods to generate systematically altered forms of CCAAT/enhancer-binding protein (C/EBP). The aim of our experiments was to identify regions of C/EBP that contribute to its capacity to activate transcription from the promoter of the serum albumin gene in cultured hepatoma cells. Earlier experiments had shown that the DNA-binding domain must remain intact for C/EBP to activate albumin transcription. We now provide evidence of two additional elements of C/EBP that are required for its gene-activating role. One such element occurs within a 28-residue region located close to the amino terminus of the protein. The other maps to a broader, more internal region of the protein and appears to exhibit functional redundancy. These newly defined elements of C/EBP exhibit two characteristics of "activation" domains delineated in studies of other gene regulatory proteins. First, they play no obvious role in the capacity of C/EBP to bind to its DNA substrate. Second, they retain function after being appended onto the DNA-binding domain of a different protein. Neither of these putative activating elements is characterized by overt distinction in either charge or preponderance of any particular amino acid. The more amino-terminal element does, however, exhibit several features suggesting that it may assume an alpha-helical structure. These studies offer observations and reagents that will be valuable for future studies concerning the physiologic function of C/EBP.
Integration host factor is required for the activation of developmentally regulated genes in Caulobacter.
Authors: Gober J W , Shapiro L
Published: June 2007

Several temporally controlled flagellar genes in Caulobacter crescentus require a sigma 54 promoter and upstream sites for transcription activation. We demonstrate here that in some of these genes, an AT-rich region containing an integration host factor (IHF) consensus binding site lies between the activator and the promoter, and that this region binds IHF in vitro. Analysis of mutations in the IHF-binding region of the hook operon demonstrated that an intact IHF-binding site is necessary for transcription in vivo. An adjacent and divergent promoter also has an IHF consensus sequence that binds IHF. The IHF and enhancer sites are 3' to the transcription start site in this promoter. We postulate that IHF mediates the formation of a higher order structure between the divergent promoter regions in a manner analogous to the nucleosome-like structure generated for lambda-Escherichia coli DNA recombination and that this higher order structure modulates transcription.
A major positive regulatory region located far upstream of the human alpha-globin gene locus.
Authors: Higgs D R , Wood W G , Jarman A P , Sharpe J , Lida J , Pretorius I M , Ayyub H
Published: June 2007

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.
The characterization of the TFIIIA synthesized in somatic cells of Xenopus laevis.
Authors: Kim S H , Darby M K , Joho K E , Brown D D
Published: June 2007

In somatic cells of Xenopus, transcription of the TFIIIA gene initiates greater than 200 bp upstream from the start site used in oocytes. The resultant mRNA encodes a protein, S-TFIIIA, that is 22 amino acids longer at its amino terminus than the abundant form of TFIIIA in oocytes (O-TFIIIA). S-TFIIIA binds the 5S RNA gene and 5S RNA, and both O- and S-TFIIIA promote the formation of stable transcription complexes on oocyte-type 5S RNA genes in an oocyte nuclear extract. We have not found any functional difference between the two forms of TFIIIA. Different transcription start sites suggest differential promoter usage--one in oocytes that permits high levels of gene activity and another that is used in somatic cells for low-level TFIIIA mRNA synthesis.
A TATA-like sequence located downstream of the transcription initiation site is required for expression of an RNA polymerase II transcribed gene.
Authors: Carcamo J , Maldonado E , Cortes P , Ahn M H , Ha I , Kasai Y , Flint J , Reinberg D
Published: June 2007

TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-like sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif.
Amino acid changes in conserved regions of the beta-subunit of Escherichia coli RNA polymerase alter transcription pausing and termination.
Authors: Landick R , Stewart J , Lee D N
Published: June 2007

Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the beta-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the beta-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the beta-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the beta-subunit are conserved between prokaryotic and eukaryotic beta-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase beta-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the beta-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.
Expression of a cAMP receptor gene of Dictyostelium and evidence for a multigene family.
Authors: Saxe C L , Johnson R L , Devreotes P N , Kimmel A R
Published: June 2007

We have previously reported the cloning of cDNAs for a Dictyostelium cell-surface cAMP receptor that is a member of the family of G-protein-linked receptors. Here, we report the organization and the developmental expression of this cAMP receptor gene, designated CAR1. CAR1 is a single copy gene that contains two intervening sequences. CAR1 mRNA levels are low in growing cells, rise to peak expression at 5-10 hr of development when the cAMP signaling system is maximally active, and decrease as development proceeds. At 5 hr the predominant mRNA species is approximately 1.9 kb, by 10 hr the mRNA is heterogeneous with sizes of approximately 1.9-2.1 kb, but during culmination only the 2.1 kb mRNA is detected. The variety of mRNA sizes results from differences in 5'-untranslated regions. Studies using developmental mutants with aberrant cAMP-signaling patterns indicate that pulsatile action of cAMP promotes maximal expression of CAR1 during early development. Low stringency hybridization of CAR1 probes to genomic DNA detects additional, related sequences, suggesting that there are several genes that encode a family of structurally similar receptors. Multiple functions previously attributed to the cAMP receptor instead may be fulfilled by distinct receptor subtypes encoded by specific genes.
Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.
Authors: Mitchell P J , Timmons P M , Hébert J M , Rigby P W , Tjian R
Published: June 2007

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.
Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene.
Authors: Cherbas L , Lee K , Cherbas P
Published: June 2007

We have identified ecdysone-response elements (EcREs) by studying regulation of the steroid-responsive Drosophila Eip28/29 gene. First, functional assays of deletion mutants identified large sequence regions required for the response; then a blotting method using the specifically labeled steroid receptor as probe identified receptor-binding regions. Three short receptor-binding regions near Eip28/29 have been identified: Prox and Dist [521 and 2295 nucleotides, respectively, downstream of the poly(A) site] are probably required for the Eip28/29 response in cell lines; Upstream (-440) is unnecessary for that response. We have also demonstrated that an EcRE-containing region from hsp27 contains a receptor-binding site. Each of these four receptor-binding regions functions as an EcRE when placed upstream of an ecdysone nonresponsive promoter and each contains an imperfect palindrome, suggesting the consensus 5'-RG(GT)TCANTGA(CA)CY-3'. Furthermore, a synthetic 15-bp fragment containing an imperfect palindrome similar to the consensus is a fully functional EcRE. The presence of any of the EcREs leads, in the absence of hormone, to depressed gene expression. When hormone is added, it relieves this repression and causes additional activation. The similarity of the EcRE sequence to response elements for estrogen, thyroid hormone, and retinoic acid receptors suggests that the steroid receptors and their signal transduction mechanisms have been strongly and broadly conserved.
Troponin I is encoded in the haplolethal region of the Shaker gene complex of Drosophila.
Authors: Barbas J A , Galceran J , Krah-Jentgens I , de la Pompa J L , Canal I , Pongs O , Ferrús A
Published: June 2007

We have analyzed one of the nine complementation groups that constitute the haplolethal (HL) region of the Shaker gene complex (ShC). Five mutations, including a dominant lethal, define this complementation group: HL I. Mutant phenotypes show abnormal embryogenesis with structural defects in the nervous system and aberrant degeneration of specific adult muscles in addition to altered action potentials. HL I encodes a family of proteins with extensive homology to invertebrate troponin I (TnI). Members of this family are brought about by two alternative and two mutually exclusive exons in conjunction with two differential polyadenylation sites. Transcription analysis indicates that some isoforms are adult specific and others are synthesized throughout development, except during early metamorphosis. Certain isoforms of Drosophila TnI are expressed in specific muscles. The specificity of mutant phenotypes suggests a functional role of particular TnI isoforms in the development and the mature activity of muscle and nervous systems.
A dissection of the cauliflower mosaic virus polyadenylation signal.
Authors: Sanfaçon H , Brodmann P , Hohn T
Published: June 2007

Mutagenesis analysis of the polyadenylation [poly(A)] signal from the cauliflower mosaic virus (CaMV), a plant pararetrovirus, revealed striking differences to known vertebrate poly(A) signals. Our results show that (1) the AATAAA sequence is necessary for efficient cleavage at the poly(A) site, although the requirement for an authentic AATAAA might be less stringent in plant than in vertebrate cells; (2) surprisingly and in contrast to the majority of vertebrate poly(A) signals, the sequences downstream of the CaMV poly(A) site do not influence processing efficiency drastically although they affect the precision of cleavage; and (3) deletion of sequences upstream of the CaMV AATAAA sequence decreased processing at the CaMV site dramatically, suggesting the presence of one or several positively acting upstream elements. An oligonucleotide consisting of CaMV upstream sequences could induce the recognition of a normally silent exogenous poly(A) signal when inserted upstream of its AATAAA motif.
In vivo UV cross-linking of U snRNAs that participate in trypanosome trans-splicing.
Authors: Watkins K P , Agabian N
Published: June 2007

The maturation of mRNAs in Trypanosoma brucei involves a trans-splicing reaction whereby the 5' 39 nucleotides of a small RNA, called the spliced leader (SL) RNA, are joined with a pre-mRNA transcript. The trans-splicing reaction appears mechanistically similar to cis-splicing of nuclear pre-mRNAs, and homologs of the U2, U4, and U6 snRNAs are required for the process. In the work presented here, potential RNA-RNA interactions between the SL RNA and the U snRNAs of trypanosomes were examined by UV light induction of RNA-RNA cross-links in vivo. We detected cross-linkage between U2 and U6 RNAs and, as might be expected, between the trypanosome U4 and U6 RNAs. The latter contain extensive sequence complementarity and are thought to exist predominantly in a single RNP. We also detected an SL RNA species following in vivo UV treatment, which may represent either an intramolecular cross-link in the SL RNA or a cross-link formed between the SL RNA and an as yet unidentified small RNA. Mapping of the cross-link position between U2 and U6 RNAs is consistent with base-pairing between the 5' domain of U2 and the 3' end of U6 RNA. These results reveal the existence, in vivo, of cognate RNA-RNA interactions in the RNA homologs that participate in trans-splicing in trypanosomes and cis-splicing in other eukaryotes.
Functional analysis of the Drosophila twist promoter reveals a dorsal-binding ventral activator region.
Authors: Pan D J , Huang J D , Courey A J
Published: June 2007

twist is one of the earliest expressed zygotically active genes required for dorsal-ventral pattern formation in the Drosophila embryo. Genetic studies suggest that this gene is activated in the ventral part of the blastoderm by maternally expressed dorsal gene product. Using P-element-mediated germ-line transformation, we have mapped a small (260 bp) dorsal-dependent ventral activator region (VAR) in the 5'-flanking region of the twist promoter that can direct the early ventral expression of a heterologous promoter. The VAR contains binding sites for a number of proteins present in extracts of Drosophila embryos. One of these sites bears homology to known binding sites for the dorsal transcription factor and is specifically bound by bacterially expressed dorsal protein. Furthermore, a 37-bp deletion that removes the dorsal-binding sequences abolishes the ventral-specific activity of the twist promoter constructs. Our data also show that additional sequences within the VAR are required to render the dorsal-binding sites functional. Finally, reverse genetic and biochemical data suggest that the transcription factor, encoded by the zeste gene may help control the overall level, if not the pattern, of twist expression.
Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on sigma 54 and modulated by positive and negative feedback mechanisms.
Authors: Weiner L , Brissette J L , Model P
Published: June 2007

The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the sigma-factor sigma 54. Negative regulation is mediated by both PspA and the sigma 32-dependent heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the sigma 32- and sigma E-dependent systems, for genes induced by heat and other stresses.
The yeast RME1 gene encodes a putative zinc finger protein that is directly repressed by a1-alpha 2.
Authors: Covitz P A , Herskowitz I , Mitchell A P
Published: June 2007

In the yeast Saccharomyces cerevisiae, a/alpha cells can enter meiosis whereas a and alpha cells cannot. The a/alpha cell type is determined by presence of a repressor, a1-alpha 2. Previous studies indicate that a/alpha cells lack an inhibitor of meiosis, the RME1 gene product, and that a and alpha cells express RME1. We report here the sequence of RME1 and functional analysis of its regulatory and coding regions. The 5'-region of RME1 includes a sequence resembling a1-alpha 2 repression sites. Deletion of this site at RME1 relieves repression by a1-alpha 2, and insertion of the site into a heterologous regulatory region (CYC1) confers weak repression in a/alpha cells. These observations indicate that RME1 is directly repressed by a1-alpha 2. The RME1 product has three regions that resemble C2H2 zinc fingers, which are characteristic of a class of nucleic-acid-binding proteins. Substitution of serine for cysteine in each of the putative fingers abolishes RME1 function; serine substitutions in the second and third putative fingers do not affect RME1 stability. These findings indicate that at least two putative zinc fingers are critical for RME1 structure or activity. Therefore RME1, which is formally a negative regulator of the meiotic gene IME1, may act directly as a repressor.
Changes in a SWI4,6-DNA-binding complex occur at the time of HO gene activation in yeast.
Authors: Taba M R , Muroff I , Lydall D , Tebb G , Nasmyth K
Published: June 2007

The yeast HO gene is transcribed transiently during G1 as cells undergo START. START-specific HO activation requires two proteins, SWI4 and SWI6, which act via a motif (CACGA4) repeated up to 10 times within the URS2 region of the HO promoter. We identified a DNA-binding activity containing SWI4 and SWI6 that recognizes the CACGA4 sequences within URS2. Two forms of SWI4,6-DNA complexes called L and U can be distinguished by their electrophoretic mobility. L complexes can be detected at all stages of the cell cycle, but U complexes are only detected in cells that have undergone START. The formation of U complexes may be the trigger of HO activation. The SWI6 protein is concentrated in the nucleus throughout G1, but at some point in S or G2 significant amounts accumulate in the cytoplasm. This change in cellular location of the SWI6 protein might contribute to the turnoff of HO transcription after cells have undergone START.
The tubB alpha-tubulin gene is essential for sexual development in Aspergillus nidulans.
Authors: Kirk K E , Morris N R
Published: June 2007

The filamentous fungus Aspergillus nidulans has two genes encoding alpha-tubulin, tubA and tubB. Mutational analysis of tubA has demonstrated that the tubA gene is essential for mitosis and nuclear migration. In this study we have deleted the tubB gene by replacing it with a selectable marker and have named this new allele tubB delta. The results demonstrate that the tubB gene is not required for vegetative growth or asexual reproduction, nor is it required for the initiation or early stages of sexual differentiation. Deletion of tubB, however, completely prevents ascosporogenesis, because tubB delta strains produce no sexual spores when self-crossed. These strains produce viable ascospores when outcrossed to tubB+ strains, indicating that the tubB delta mutation is recessive. We have studied the cytology of sexual development in wild-type strains and in the tubB mutant and have observed that tubB delta. strains develop normally to the stage of ascus formation. However, only a single nuclear mass is observed in the tubB delta ascus, indicating that either the two zygotic haploid nuclei are blocked in karyogamy or that karyogamy occurs but the resulting diploid nucleus is subsequently blocked in meiosis I.
Homologous recombination in the tandem calmodulin genes of Trypanosoma brucei yields multiple products: compensation for deleterious deletions by gene amplification.
Authors: Eid J E , Sollner-Webb B
Published: June 2007

Homologous recombination between a calmodulin-neomycin-resistance fusion gene and the Trypanosoma brucei chromosome takes place not only in the large 5'- and 3'-flanking segments of the calmodulin locus but also in any of the four tandem genomic calmodulin genes. This results in a recombined locus consisting of the chimeric neor gene and four, three, two, one, or zero functional calmodulin genes. Cells bearing this latter event have half of their normal number of intact calmodulin genes and an accompanying phenotype of slow growth. Over months of propagation, these lines acquire additional calmodulin genes, frequently by amplifying a calmodulin gene at the untargeted locus, and concomitantly revert to normal growth rate. This response could be related to the property of the trypanosome of maintaining most housekeeping genes in tandem chromosomal arrays. Recombination appears to be initiated by a crossover event between the linearized end of the transfecting plasmid and a homologous region in the host genome; the second crossover generally occurs internally and in that region requires no more than 87 bp of homology.
v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.
Authors: Disela C , Glineur C , Bugge T , Sap J , Stengl G , Dodgson J , Stunnenberg H , Beug H , Zenke M
Published: June 2007

The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein.
Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression.
Authors: Whiting J , Marshall H , Cook M , Krumlauf R , Rigby P W , Stott D , Allemann R K
Published: June 2007

Murine Hox genes are organized into four clusters that share many features with the homeotic clusters of Drosophila. This evolutionary conservation and the clear relationships between the position of a gene within a cluster and its expression pattern have led to the suggestion that the structure of the cluster is essential for proper regulation. Using a Hox-2.6-lacZ reporter gene in transgenic mice we have shown that the overall expression pattern of the endogenous Hox-2.6 gene can be reconstructed when it is isolated from the complex. The transgene was expressed in the proper tissues, with the correct spatial distribution and temporal pattern. Furthermore, direct comparison by in situ hybridization revealed that the levels of transgene expression are similar to those of the endogenous gene. This has allowed us to define three elements that regulate particular aspects of the Hox-2.6 pattern, two of which act as spatially specific enhancers. One enhancer, region A, directed expression only in the neural tube, whereas the other, region C, specified the majority of the Hox-2.6 pattern. Both were also capable of imposing the correct boundaries of expression on heterologous promoters. The definition of such elements will allow the characterization of the trans-acting factors that mediate spatial regulation in the mammalian embryo.
tudor, a posterior-group gene of Drosophila melanogaster, encodes a novel protein and an mRNA localized during mid-oogenesis.
Authors: Golumbeski G S , Bardsley A , Tax F , Boswell R E
Published: June 2007

The tudor (tud) locus of Drosophila melanogaster is required during oogenesis for the formation of primordial germ cells and for normal abdominal segmentation. The tud locus was cloned, and its product was identified by Northern analysis of wild-type and tud mutant RNAs. The locus encodes a single mRNA of approximately 8.0 kb that is expressed throughout the life cycle, beginning in the early stages of germ-line development in the female. During oogenesis, tud mRNA appears to be present in the oocyte precursor within the germarial cysts, and in stages 1-3 it accumulates within the developing oocyte. The transcript is localized to the posterior half of the oocyte during oogenetic stages 4-7 but is not detectable within the ooplasm by egg deposition and throughout early embryogenesis. The tud protein has a predicted molecular mass of 285,000 daltons and has no distinctive sequence similarity to known proteins or protein structural motifs. Taken together, these results indicate that the tud product is a novel protein required during oogenesis for establishment of a functional center of morphogenetic activity in the posterior tip of the Drosophila embryo.
Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation.
Authors: Ryner L C , Baker B S
Published: June 2007

Sex-specific alternative processing of the doublesex (dsx) pre-mRNA controls somatic sexual differentiation in Drosophila melanogaster. Processing in the female-specific pattern results from the utilization of an upstream 3'-terminal exon and requires the activities of both the transformer (tra) and transformer-2 (tra-2) genes. Use of the more downstream male-specific terminal exons does not require the activities of these genes and is thus considered the default dsx-processing pattern. Here, we used transient expression of dsx pre-mRNAs in the presence or absence of tra and tra-2 gene products in Drosophila tissue culture cells to investigate the molecular mechanism controlling this alternative RNA-processing decision. These studies reveal that female-specific processing of dsx pre-mRNA is controlled by tra and tra-2 through the positive regulation of female-specific alternative 3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a 540-nucleotide region from within the female exon is both necessary and sufficient for regulation. In addition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently of female-specific polyadenylation. Regulated polyadenylation was obtained only in the presence of splicing, suggesting that activation of female-specific exon use occurs by 3'SS activation.
Attenuation of the heat shock response in HeLa cells is mediated by the release of bound heat shock transcription factor and is modulated by changes in growth and in heat shock temperatures.
Authors: Abravaya K , Phillips B , Morimoto R I
Published: June 2007

When HeLa S3 cells are subjected to a continuous 42 degrees C heat shock, activation of heat shock transcription factor (HSF) and transcriptional activation of the heat shock genes hsp70, hsp89 alpha, and hsp60 is transient, peaking at 40-60 min of heat shock, and then attenuating. We have used in vivo genomic footprinting to demonstrate that attenuation of hsp70 transcription is mediated by release of bound HSF from the heat shock element (HSE) of the hsp70 gene promoter. Release of bound HSF in vivo occurs at a higher rate than would be predicted from in vitro measurements of dissociation. Attenuation of HSF activation and heat shock gene transcription occurs only when mild heat shock temperatures are employed (42 degrees C); increasing the heat shock temperature by 1 degree C elicits a much higher level of activation, which does not attenuate during a 4-hr heat shock. Surprisingly, altering the temperature at which cells are grown prior to heat shock modulates the magnitude and temporal pattern of the response to a given heat shock temperature. This finding suggests that HSF does not sense temperature directly but, instead, may be responsive to the magnitude of the difference between growth and heat shock temperatures.
Retinoic acid modifies mesodermal patterning in early Xenopus embryos.
Authors: Ruiz i Altaba A , Jessell T
Published: June 2007

Treatment of early Xenopus embryos with retinoic acid (RA) produces a concentration-dependent series of defects in anterior axial structures that range from small deletions to embryos lacking heads. The graded series of axial defects obtained after RA administration to early embryos appears to result, in part, from actions of RA on embryonic mesoderm. RA modifies the differentiation of anterior dorsal mesoderm from animal cap ectoderm induced by mesoderm-inducing peptide growth factors (PGFs). Concentrations of RA that suppress anterior dorsal mesoderm result in the differentiation of mesoderm of more posterior or ventral character. The suppression of anterior dorsal mesoderm may account for the absence of anterior neural ectoderm after RA treatment. Although RA changes the character of mesoderm, it does not seem to affect mesodermal induction by PGFs or the levels of Xhox3 mRNA induced in the mesoderm by PGFs. RA therefore appears to affect steps downstream from those involved in the initial induction of mesoderm. In experiments to examine the possible physiological role of RA in early Xenopus development, dorsal and ventral ectoderm were found to respond differently to identical concentrations of PGFs. One potential basis for this heterogeneity is the existence of a localized inhibitor, possibly RA, in the early Xenopus embryo. RA could therefore contribute to axial patterning by inhibiting the development of mesoderm of different character induced by PGFs.