bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle.
Authors: Shieh J C , Wilkinson M G , Buck V , Morgan B A , Makino K , Millar J B
Published: June 2007

The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK. We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.
A shared subunit belongs to the eukaryotic core RNA polymerase.
Authors: Lanzendörfer M , Smid A , Klinger C , Schultz P , Sentenac A , Carles C , Riva M
Published: June 2007

The yeast RNA polymerase I is a multimeric complex composed of 14 distinct subunits, 5 of which are shared by the three forms of nuclear RNA polymerase. The reasons for this structural complexity are still largely unknown. Isolation of an inactive form of RNA Pol I lacking the A43, ABC23, and A14 subunits (RNA Pol I delta) allowed us to investigate the function of the shared subunit ABC23 by in vitro reconstitution experiments. Addition of recombinant ABC23 alone to the RNA Pol I delta reactivated the enzyme to up to 50% of the wild-type enzyme activity. The recombinant subunit was stably and stoichiometrically reassociated within the enzymatic complex. ABC23 was found to be required for the formation of the first phosphodiester bond, but it was not involved in DNA binding by RNA Pol I, as shown by gel retardation and surface plasmon resonance experiments, and did not recycle during transcription. Electron microscopic visualization and electrophoretic analysis of the subunit depleted and reactivated forms of the enzyme indicate that binding of ABC23 caused a major conformational change leading to a transcriptionally competent enzyme. Altogether, our results demonstrate that the ABC23 subunit is required for the structural and functional integrity of RNA Pol I and thus should be considered as part of the core enzyme.
Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development.
Authors: Yan Y , Frisén J , Lee M H , Massagué J , Barbacid M
Published: June 2007

p57(Kip2) is a paternally imprinted gene that encodes a potent inhibitor of several cyclin/Cdk complexes. p57(Kip2) is primarily expressed in terminally differentiated cells, associates with G1 Cdks, and can cause cell cycle arrest in G1 phase. To investigate the role of p57(Kip2) in vivo, we have ablated the p57(Kip2) gene by homologous recombination in ES cells and generated mice devoid of p57(Kip2) expression. Most p57(Kip2) null mice die after birth and display severe developmental defects with varying degrees of penetrance. As expected, heterozygous mice that inherit a maternal, but not a paternal, targeted allele exhibit similar deficiencies and neonatal death. Developmental defects of p57(Kip2) mutant mice include cleft palate and gastrointestinal abnormalities ranging from an inflated GI tract to loss of the jejunum and ileum. These tissues display a significant increase of apoptotic cells in the absence of p57(Kip2). Most p57(Kip2) mutant mice have short limbs, a defect attributable to abnormal endochondral ossification caused by delayed cell cycle exit during chondrocyte differentiation. A similar defect has been observed in mice lacking p107 and p130, thus suggesting that p57(Kip2) might be an upstream regulator of these Rb-related proteins. The p57(Kip2) locus has been implicated in the Beckwith-Wiedemann syndrome and in the development of sporadic Wilms' tumors and lung carcinomas. To date, we have not observed neoplastic development even in those p57(Kip2) mutant mice that have survived for >5 months of age. These findings indicate that p57(Kip2) has an important role during mouse development that cannot be compensated by other Cdk inhibitors.
The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase.
Authors: Kretzschmar M , Liu F , Hata A , Doody J , Massagué J
Published: June 2007

Bone morphogenetic proteins (BMPs) are members of the TGF-beta family that regulate cell proliferation, apoptosis, and differentiation, and participate in the development of most tissues and organs in vertebrates. Smad proteins function downstream of TGF-beta receptor serine/threonine kinases and undergo serine phosphorylation in response to receptor activation. Smad1 is regulated in this fashion by BMP receptors, and Smad2 and Smad3 by TGF-beta and activin receptors. Here, we report that BMP receptors phosphorylate and activate Smad1 directly. Phosphorylation of Smad1 in vivo involves serines in the carboxy-terminal motif SSXS. These residues are phosphorylated directly by a BMP type I receptor in vitro. Mutation of these carboxy-terminal serines prevents several Smad1 activation events, namely, Smad1 association with the related protein DPC4, accumulation in the nucleus, and gain of transcriptional activity. Similar carboxy-terminal serines in Smad2 are required for its phosphorylation and association with DPC4 in response to TGF-beta, indicating the generality of this process of Smad activation. As a direct physiological substrate of BMP receptors, Smad1 provides a link between receptor serine/threonine kinases and the nucleus.
DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.
Authors: Campbell K S , Mullane K P , Aksoy I A , Stubdal H , Zalvide J , Pipas J M , Silver P A , Roberts T M , Schaffhausen B S , DeCaprio J A
Published: June 2007

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.
Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase.
Authors: Sung P
Published: June 2007

Saccharomyces cerevisiae RAD51, RAD55, and RAD57 genes, required for genetic recombination and DNA double-strand-break repair, encode proteins homologous to one another and to the Escherichia coli RecA protein. Rad51 protein catalyzes the DNA strand-exchange reaction with a dependence on ATP and on the heterotrimeric single-strand DNA (ssDNA) binding factor replication protein A (RPA). By several independent criteria, RAD55- and RAD57-encoded products are shown here to exist as a stable heterodimer, with a dissociation constant of <2 x 10(-10) M. In strand exchange, the reaction proceeds efficiently if RPA is incorporated after nucleation of Rad51 onto ssDNA, but if RPA is present during the nucleation phase, as is likely the case in vivo, the amount of strand-exchange products becomes relatively insignificant. Inclusion of the Rad55-Rad57 heterodimer with Rad51 and RPA results in a marked stimulation of strand exchange, providing evidence for a role of the Rad55-Rad57 heterodimer in overcoming the inhibitory effect of RPA.
14-3-3 epsilon positively regulates Ras-mediated signaling in Drosophila.
Authors: Chang H C , Rubin G M
Published: June 2007

We have isolated mutations in the gene encoding a Drosophila 14-3-3 epsilon protein as suppressors of the rough eye phenotype caused by the ectopic expression of RAS1(V12). Using a simple loss-of-function 14-3-3 epsilon mutation, we show that 14-3-3 epsilon acts to increase the efficiency of RAS1 signaling. The 14-3-3 epsilon protein appears to function in multiple RTK pathways, suggesting that it is a general component of RAS1 signaling cascade. Sequence analysis of three dominant-negative alleles defines two regions of 14-3-3 epsilon that participate in RAS1 signaling. We also present evidence that 14-3-3 epsilon and 14-3-3 zeta, two 14-3-3 protein family members, are partially redundant for RAS1 signaling in photoreceptor formation and in animal viability. Our genetic data suggest that 14-3-3 epsilon functions downstream of or parallel to RAF, but upstream of nuclear factors in RAS1 signaling.
The sigma(E) and the Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli.
Authors: Danese P N , Silhavy T J
Published: June 2007

In Escherichia coli, the heat shock-inducible sigma-factor sigma(E) and the Cpx two-component signal transduction system are both attuned to extracytoplasmic stimuli. For example, sigma(E) activity rises in response to the overproduction of various outer-membrane proteins. Similarly, the activity of the Cpx signal transduction pathway, which consists of an inner-membrane sensor (CpxA) and a cognate response regulator (CpxR), is stimulated by overproduction of the outer-membrane lipoprotein, NlpE. In response to these extracytoplasmic stimuli, sigma(E) and CpxA/CpxR stimulate the transcription of degP, which encodes a periplasmic protease. This suggests that CpxA/CpxR and sigma(E) both mediate protein turnover within the bacterial envelope. Here, we show that CpxA/CpxR and sigma(E) also control the synthesis of periplasmic enzymes that can facilitate protein-folding reactions. Specifically, sigma(E) controls transcription of fkpA, which specifies a periplasmic peptidyl-prolyl cis/trans isomerase. Similarly, the Cpx system controls transcription of the dsbA locus, which encodes a periplasmic enzyme required for efficient disulfide bond formation in several extracytoplasmic proteins. Taken together, these results indicate that sigma(E) and CpxA/CpxR are involved in regulating both protein-turnover and protein-folding activities within the bacterial envelope.
Spatially deranged though temporally correct expression of Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family.
Authors: Franks R R , Hough-Evans B R , Britten R J , Davidson E H
Published: June 2007

We report the unexpected observation that cis-regulatory sequences of a Strongylocentrotus purpuratus actin gene, which direct a particular, lineage-specific pattern of embryonic expression, confer a completely different spatial pattern of expression when introduced into embryos of another sea urchin species. We utilized a fusion gene construct in which the bacterial chloramphenicol acetyl transferase (CAT) reporter gene is driven by CyIIIa actin regulatory sequences. We previously showed that the regulatory region that is included suffices to promote the accumulation of CAT mRNA in transgenic S. purpuratus embryos, on the same developmental schedule and in the same embryonic region, the aboral ectoderm, in which the CyIIIa actin gene is normally expressed (Flytzanis et al. 1987; Hough-Evans et al. 1987). When injected into zygotes of Lytechinus variegatus, which belongs to a different echinoid family, the expected temporal pattern of expression of CAT enzyme was observed. Thus, in both S. purpuratus and L. variegatus embryos, expression is activated at the early blastula stage, although this stage is attained several hours sooner in L. variegatus embryo cultures. Similar kinetics of CAT enzyme accumulation were obtained whether the gene was introduced directly into the L. variegatus zygote nucleus or into the cytoplasm. However, when examined by in situ hybridization, the transgenic L. variegatus embryos were found to display a totally new pattern of CAT mRNA accumulation. Copious CAT transcripts were detected not only in aboral ectoderm cells, but also in skeletogenic mesenchyme cells, gut cells, and oral ectoderm, all cell types that in the transgenic S. purpuratus controls are invariably devoid of detectable CAT transcripts.
Limited transcription of rat elastase I transgene repeats in transgenic mice.
Authors: Davis B P , MacDonald R J
Published: June 2007

The rat elastase I (EI) regulatory region is an enhancer that directs efficient pancreas-specific transcription of linked genes integrated in the chromosomes of transgenic mice. However, with increasing numbers of tandemly repeated EI transgenes, the transcription rate per gene decreased. This decrease was not due to a titration of essential transcription factors, because transgenic mice with as many as 250 copies of the rat EI enhancer continued transcription of the endogenous mouse EI gene at its normal rate. Furthermore, when transgenic mice bearing 250 EI genes and mice bearing 7 copies of an EI enhancer-directed human growth hormone (hGh) gene were mated to produce mice with two unlinked arrays of EI enhancer-driven transgenes, each array was transcribed at the same rate as in mice bearing each array separately. Long tandem arrays of transgenes may inhibit efficient transcription despite the presence of ample amounts of essential transcription factors. Although the transcription rate of the mouse EI gene was not affected by the presence of large numbers of transgenes, the amounts of mouse elastase I and elastase II mRNAs were decreased in the presence of high levels of transgene mRNA, indicating an adjustment of the cell mRNA population.
In vitro transcription of the Drosophila engrailed gene.
Authors: Soeller W C , Poole S J , Kornberg T
Published: June 2007

An enzyme system that accurately initiates transcription of the engrailed gene has been prepared from Drosophila embryos. The system has been separated chromatographically into two fractions, both of which are required for specific engrailed transcription. DNase footprint and competition analysis detected at least two sequence-specific DNA-binding proteins in one of these two fractions. Together, these proteins bind to eight regions within 400 bp of the transcription initiation sites. Most of the regions containing these binding sites are required for manimal engrailed transcription in vitro. In addition, a region downstream from the initiation sites and within the first 40 residues of the transcription unit is essential for transcription. Transient in vivo expression assays indicated that these same upstream and downstream sequences are required for transcription in Drosophila tissue culture cells.
Interacting genes that affect microtubule function: the nc2 allele of the haywire locus fails to complement mutations in the testis-specific beta-tubulin gene of Drosophila.
Authors: Regan C L , Fuller M T
Published: June 2007

A mutation that fails to complement certain alleles of the testis-specific beta 2-tubulin gene (B2t) of Drosophila melanogaster maps to a separate locus, haywire, located at 3-34.4 map units in polytene region 67E3-F3. Second-site non-complementing mutations such as haync2 and B2t alleles could identify genes that encode products that participate in the same functions or that interact in the same structure. Consistent with a structural interaction between the hay gene product and beta 2-tubulin, the genetic interaction between haync2 and B2t requires the presence of the mutant hay gene product; a deficiency for the hay region complements the same alleles of B2t that haync2 fails to complement. haync2 is a recessive male sterile mutation in a genetic background that is wild type at the B2t locus. Homozygous males have defects in meiosis, flagellar elongation and nuclear shaping, the three major microtubule-based processes in which the testis-specific beta 2-tubulin participates. The haync2 allele also has effects outside of spermatogenesis. It is a temperature-sensitive semilethal mutation, and homozygous haync2 females have reduced fertility. These phenotypes are consistent with a role for the haywire gene product in general microtubule function. Analysis of second-site non-complementing mutations such as haync2 offers a genetic tool for analysis of interacting proteins in complex assemblies.
A family of immunologically related transcription factors that includes multiple forms of ATF and AP-1.
Authors: Hai T W , Liu F , Allegretto E A , Karin M , Green M R
Published: June 2007

ATF is a cellular transcription factor involved in the regulation of multiple adenovirus E1A- and cellular cAMP-inducible promoters. Using DNA affinity chromatography, we have purified ATF and found that a series of polypeptides copurify in a sequence-specific manner. We demonstrate that these polypeptides represent a family of proteins that are related by DNA-binding specificity and by immunological cross-reactivity. This family includes the transcription factor AP-1, whose recognition sequence, GTGAGTCAA, differs from the ATF consensus, GTGACGTCAA, by the absence of a cytosine residue. Our results further indicate that there are multiple forms of both ATF and AP-1. The immunological cross-reactivity and related DNA-binding specificities suggest that ATF and AP-1 contain similar amino acid sequences and may have originated from a common gene.
Suppression of SV40-promoted gene expression by differentiation of preadipose cells.
Authors: Djian P , Phillips M , Green H
Published: June 2007

When a plasmid bearing the chloramphenicol acetyltransferase (CAT) gene under the control of an SV40 early promoter is introduced into preadipose or adipose cells of line 3T3-F442A, the promoter directs high levels of transient expression of CAT. However, when the plasmid is introduced into preadipose cells and the cells are then allowed to differentiate into adipose cells, the expression of the CAT gene is suppressed. In this process, the plasmid is not changed detectably in amount, topology, or state of methylation. Stably transformed preadipose cells bearing an integrated plasmid express the transferase, but if the cells are allowed to differentiate, the expression of the gene is similarly suppressed. The decline in CAT activity is associated with a decrease in the transcription rate of the gene. Transcription of a gene coding for neomycin phosphotransferase driven by the SV40 promoter is also greatly diminished by differentiation. Because suppression of CAT does not occur when the gene is under control of a retroviral long terminal repeat (LTR), a specific mechanism exists for the recognition and inactivation of the SV40 early promoter during differentiation.
Destruction of a translationally controlled mRNA in Xenopus oocytes delays progesterone-induced maturation.
Authors: Smith R C , Dworkin M B , Dworkin-Rastl E
Published: June 2007

The maternal mRNA D7 is a moderately abundant transcript in Xenopus laevis whose expression is highest in, and perhaps restricted to, oogenesis and early embryogenesis. The nucleotide sequence of cloned D7 cDNA was determined and shown to have the capacity to code for a 31-kD protein. This amino acid sequence was searched against a protein data base, and no homologous proteins were found. Antibodies directed against D7 recognize in Xenopus embryos a soluble, cytoplasmic protein with an apparent molecular weight on SDS gels of 36,000. The D7 protein is absent from oocytes and first begins to accumulate during oocyte maturation. Its levels are highest during the first day of embryonic development and then decrease; D7 protein was not detected in adult tissues. D7 mRNA was selectively destroyed by injection into oocytes of antisense oligodeoxynucleotides. Analysis of injected oocytes by Northern and Western blotting showed site-specific cleavage and subsequent degradation of the D7 mRNA and the failure of the D7 protein to accumulate during progesterone-induced maturation. The loss of D7 protein affects the maturation process itself, significantly delaying the time course of germinal vesicle breakdown. Thus, D7 is a newly described protein involved in oocyte maturation.
Homologous integration in mammalian cells without target gene selection.
Authors: Jasin M , Berg P
Published: June 2007

Homologous integrations into a nonselectable target locus have been highly enriched for following DNA transfections into mammalian cells. The target gene, the SV40 early region in COS1 cells, provides transcription signals to activate a defective selectable marker, the gpt gene. We find that nearly half of the selected clones have integrated the gpt gene at the homologous sequence in the COS1 genome. This is an estimated 100-fold enrichment for homologous events compared with transfections in which the gpt gene is transcriptionally active. As shown for yeast integration events, a double-strand break at a position of homology between the transfected DNA and the genomic target is necessary to achieve a high frequency of homologous integrations. Furthermore, the arrangement of sequences at the integration site includes a repair of the double-strand gap, which was present on the transfected DNA, suggesting that similarities exist between yeast and mammalian integrations. The experimental design, in which a defective marker is activated following a homologous integration, may have general applications for gene targeting in mammalian cells.
Transcription analysis of a human U4C gene: involvement of transcription factors novel to snRNA gene expression.
Authors: Weller P , Bark C , Janson L , Pettersson U
Published: June 2007

We have investigated the promoter requirements for in vivo transcription of a human U4C snRNA gene following transfection into HeLa cells. Two elements required for maximal U4C transcription were identified. The first, located upstream of -50, provides a basal level of transcription 2-3% of the full activity, and probably corresponds to the previously identified snRNA gene proximal element. The distal element, centered around -220, acts as a transcriptional enhancer and contains motifs for three previously recognized transcription factors: the octamer-binding protein, NF-A, which binds to motifs in the distal elements of other snRNA genes, and two factors not previously shown to be involved in snRNA gene transcription, cAMP response element binding protein (CREB) and AP-2. The octamer and putative AP-2 motifs are required for maximal transcription of the U4C gene. Specific binding of NF-A and CREB to the motifs in the distal element has been shown in vitro by DNase I and DMS methylation protection footprint competition analyses using HeLa nuclear extracts. The presence of a binding motif for the inducible factor CREB, together with the transcriptional requirement for the putative AP-2 motif, suggests a means by which expression of snRNA genes might be regulated.
OBP100 binds remarkably degenerate octamer motifs through specific interactions with flanking sequences.
Authors: Baumruker T , Sturm R , Herr W
Published: June 2007

We have used the 100-kD HeLa cell octamer-binding protein OBP100 as a model to study flexible DNA sequence recognition by promoter-binding proteins. OBP100 binds to the conserved octamer motif ATGCAAAT found in numerous promoters and additionally to two degenerate octamer motifs (sites I and II) within the SV40 enhancer region. We show here that OBP100 binds the herpes simplex virus immediate early promoter TAATGARAT (R = purine) motif itself, extending the flexibility of OBP100 sequence recognition to sequences that bear very little resemblance (four matches over a 14-bp region). Nevertheless, a progression of OBP100-binding sites can be established that links the sequences of these two apparently unrelated binding sites by incremental steps. Mutational and chemical modification interference analyses of a degenerate octamer binding site (SV40 site II) show that specific sequences, which are not normally conserved but flank the degenerate octamer motif, can compensate for the degeneracy in the octamer core sequence. Thus, different regions of the binding site sequence (core or flanking) can diverge separately but not independently of one another. These results suggest that flexible DNA sequence recognition arises because there are few obligatory contact sites for OBP100 binding, but, rather, specific binding reflects the sum of many independent interactions.
The Drosophila melanogaster suppressor of Hairy-wing protein binds to specific sequences of the gypsy retrotransposon.
Authors: Spana C , Harrison D A , Corces V G
Published: June 2007

Mutations at the suppressor of Hairy-wing [su(Hw), 3-54.8] locus reverse the phenotype of second-site mutations induced by the gypsy transposable element in Drosophila melanogaster. This gene encodes a protein with a predicted molecular weight of 109,000 that contains an acidic domain and 12 copies of the DNA-binding 'Zn finger' motif. The su(Hw) protein was overexpressed in Escherichia coli and Drosophila cells, and partially purified. It was shown to interact specifically in vitro with a 367-bp DNA fragment that contains 12 copies of the sequence PyPuTTGCATACCPy located in the 5'-untranslated region of gypsy, between the 5' long terminal repeat (LTR) and the first ATG initiation codon. This sequence shows striking homology to some mammalian transcriptional enhancer elements, supporting a role for the su(Hw) protein in the control of gypsy transcription. In addition, the su(Hw) protein is present at approximately 100-200 sites on Drosophila polytene chromosomes, suggesting that it also interacts in vivo with DNA and might be involved functionally in the regulation of normal cellular genes.
Determinants of the 3' splice site for self-splicing of the Tetrahymena pre-rRNA.
Authors: Price J V , Cech T R
Published: June 2007

Tetrahymena preribosomal RNA undergoes self-splicing in vitro. The structural components involved in recognition of the 5' splice site have been identified, but the mechanism by which the 3' splice site is recognized is not established. To identify some components of 3'splice site recognition, we have generated mutations near the 3' splice site and determined their effects on self-splicing. Alteration of the 3'-terminal guanosine of the intervening sequence (IVS), a conserved nucleotide in group I IVSs, almost eliminated 3' splice site activity; the IVS-3' exon splicing intermediate accumulated, and exon ligation was extremely slow. These mutations do not result in recruitment of cryptic 3' splice sites, in contrast to mutations that affect the 5' splice site. Alteration of the cytidine preceding the 3'-terminal guanosine or of the first two nucleotides of the 3' exon had similar but less severe effects on exon ligation. Most of the mutants showed some reduction (less than threefold) in GTP addition at the 5' splice site. A mutation that placed a new guanosine residue just upstream from the 3'-terminal guanosine misspliced to produce ligated exons with one extra nucleotide between the 5' and 3' exons. We conclude that multiple nucleotides, located both at the 3' end of the IVS and in the 3' exon, are required for 3' splice site recognition.
Correct in vivo splicing of the mouse immunoglobulin kappa light-chain pre-mRNA is dependent on 5' splice-site position even in the absence of transcription.
Authors: Kedes D H , Steitz J A
Published: June 2007

In transcripts from the rearranged mouse immunoglobulin kappa light-chain locus, the intron separating the variable (V) plus joining (J) exon from the constant (C) exon contains up to three additional J regions, each with a functional 5' splice site. Previously, HeLa cells transfected with DNA encoding kappa light chains have been shown to mimic kappa-producing lymphocytes in splicing exclusively to the upstream-most 5' splice site, whereas selectivity is lost when kappa transcripts containing two more J regions are incubated in HeLa cell or lymphocyte nuclear extracts. Here we demonstrate that the fidelity of in vivo splicing depends on neither V-J rearrangement, the instability of erroneously splicing transcripts, nor a hierarchy of J-region 5' splice site utilization. Analysis of the splicing of presynthesized kappa transcripts injected into Xenopus oocytes demonstrates the correct 5' splice-site selection is independent of transcription. Implications for in vitro studies of regulated splice-site pairing are discussed.
The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity.
Authors: Sung P , Prakash S , Prakash L
Published: June 2007

The RAD6 gene of the yeast Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, DNA damage-induced mutagenesis, and sporulation. Here we demonstrate that the protein encoded by the RAD6 gene, previously shown to be a ubiquitin-conjugating (E2) enzyme, multiply ubiquitinates histones H2A and H2B efficiently to give products containing as many as seven or more molecules of ubiquitin. We also show that the highly acidic 23-residue RAD6 carboxy-terminal tail domain, which contains a total of 20 acidic residues, is essential for the histone-polyubiquitinating activity. Because the RAD6 polyacidic tail is required for the sporulation function but not for the DNA repair and induced mutagenesis functions of RAD6, the present observations suggest that the histone-polyubiquitinating activity of RAD6 protein is essential for sporulation but not for DNA repair and induced mutagenesis. Attachment of multiple molecules of ubiquitin to histones by RAD6 protein may serve to target the histones for degradation via the ubiquitin-dependent proteolytic system or to alter chromatin structure. The in vitro system for synthesizing polyubiquitinated histones described herein provides a means for investigating these possibilities.
Ha-ras and c-myc oncogene expression interferes with morphological and functional differentiation of mammary epithelial cells in single and double transgenic mice.
Authors: Andres A C , van der Valk M A , Schönenberger C A , Flückiger F , LeMeur M , Gerlinger P , Groner B
Published: June 2007

We studied the effects of the tissue-specific and lactogenic hormone-dependent expression of the recombinant whey acidic protein (Wap)-ras and Wap-myc oncogenes on the differentiation of the mammary epithelium in transgenic mice. The histological appearance of mammary glands in pregnant, lactating, and postlactational animals and their ability to express milk protein genes were analyzed. Activated Ha-ras expression caused a decrease of milk protein synthesis during the lactation period. The formation of glandular epithelium and the postlactational regression of epithelial cells were not affected. c-myc expression impaired the development of the glandular epithelium, and milk protein synthesis was decreased strongly. Epithelial cell proliferation continued during lactation and postlactationally. Coexpression of both oncogenes in double transgenic mice synergistically affected differentiation and resulted in a high number of neoplastic foci. Palpable tumors were observed only after a latency of 3-4 months. Tumor cells utilize the Wap promoter hormone independently, express increased levels of Wap-ras and induce adjacent stromal cells to produce tenascin.
A group of genes required for pattern formation in the ventral ectoderm of the Drosophila embryo.
Authors: Mayer U , Nüsslein-Volhard C
Published: June 2007

Mutations in the genes spitz (spi), Star (S), single-minded (sim), pointed (pnt), rhomboid (rho) (all zygotic), and sichel (sic) (maternal), collectively called the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects were found in the median part of the central nervous system in whole-mount embryos stained with anti-HRP (horseradish peroxidase) antibodies. In addition, the nerve cells expressing the even-skipped protein appeared abnormally arranged. These results suggest that groups of cells from the same region, including both epidermal and neural precursor cells, require spitz-group gene activity for normal development. The members of the spitz group differ from one another: sim affects a more median strip of the ventral ectoderm than the other zygotic genes and pnt causes separation rather than deletion of pattern elements. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line, while sim, rho, and pnt appear to be exclusively zygotically expressed, and the maternal gene sic acts in the germ line autonomously. Some embryos produced by sic-homozygous females differentiate the spitz phenotype, others develop normally or die early. Of all the spitz-group genes, sim appears to have the most specific effect on the embryonic pattern. The significance of the spitz-group phenotypes for the dorso-ventral pattern formation is discussed.
Tn7 transposition: two transposition pathways directed by five Tn7-encoded genes.
Authors: Waddell C S , Craig N L
Published: June 2007

The bacterial transposon Tn7 is capable of high-frequency transposition to a specific site in the Escherichia coli chromosome, attTn7, and of low-frequency transposition to sites other than attTn7. Using an in vitro insertional mutagenesis procedure, we have identified and characterized five tns (Tn seven) genes that are essential for Tn7 transposition. Three of these genes, tnsA, tnsB, and tnsC, are required, but are not sufficient, for all Tn7 transposition events. In addition, tnsD is specifically required for transposition to attTn7, whereas tnsE is specifically required for transposition to other sites. Thus, Tn7 is an elaborate transposon that encodes two distinct but overlapping transposition pathways.
Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used.
Authors: Kunkel G R , Pederson T
Published: June 2007

U6 small nuclear RNA is transcribed by a different polymerase than U1-U5 RNAs, likely to be RNA polymerase III. Transcription from human U6 gene deletion-substitution templates in a HeLa S100 extract delineated the 5' border of a control element lying between 67 and 43 bp upstream from the initiation site. This region matches the location of, and shows considerable sequence similarity with, the proximal control element of U1 and U2 RNA genes, which are transcribed by RNA polymerase II. Transfection of human 293 cells with 5'-flanking deletion-substitution mutants of a U6 maxigene revealed a dominant control element between 245 and 149 bp upstream of the transcription start site. An octamer motif was found in this region in an inverted orientation relative to that of the human U1 and U2 RNA gene enhancers but in the same orientation as a human U4 RNA gene, the transcript of which functions together with U6 RNA in a single small nuclear ribonucleoprotein (snRNP) particle. The human U2 gene enhancer joined to the U6 maxigene was able to functionally replace the U6 distal control element(s).
Expression of a collagen gene in mesenchyme lineages of the Strongylocentrotus purpuratus embryo.
Authors: Angerer L M , Chambers S A , Yang Q , Venkatesan M , Angerer R C , Simpson R T
Published: June 2007

We have previously described cloning of an exon of a sea urchin collagen gene and shown that its expression is temporally regulated during embryogenesis, beginning during blastula formation. We have now localized the protein encoded by the gene and the sites of its mRNA synthesis in the developing embryo. Antibody to a synthetic peptide reacts with a 208,000 Mr protein that is digestible by collagenase. Fractionation of pluteus stage embryos demonstrates that the protein is localized primarily with cells that form the syncytium of primary mesenchyme that elaborates the larval endoskeleton; furthermore, immunofluorescence localizes the epitope to the periphery of the endoskeleton in situ. Transcripts of the gene accumulate only in mesenchyme cells, especially those of the primary mesenchyme lineage. Measurements of absolute transcript abundance show that collagen mRNA is present in blastula primary mesenchyme cells at 600-700 copies per cell and at about fourfold lower amounts in other mesenchyme cells.
Expression of human alpha 1-acid glycoprotein genes in cultured cells and in transgenic mice.
Authors: Dente L , Rüther U , Tripodi M , Wagner E F , Cortese R
Published: June 2007

The human genome contains three alpha 1-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human alpha 1-acid glycoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regulation of the cloned AGP-A gene by transfection into cell lines of hepatic and nonhepatic origin. Unlike any other liver-specific gene investigated so far, every AGP construct tested was expressed with comparable efficiency in hepatoma and HeLa cells. In contrast, identical constructs in transgenic mice are expressed in a tissue-specific manner and are regulated by acute-phase stimuli. Transgenic mice carrying the cluster of three AGP genes secrete the human protein in the serum, and the corresponding mRNA is mainly derived from the AGP-A gene. The mRNA is liver specific, and its concentration increases several fold following experimentally induced inflammation. Additional transgenic lines carrying only the AGP-A gene showed that sufficient information for tissue-specific and regulated expression is contained within a 6.6-kb segment comprising the whole coding region plus 1.2-kb 5'-flanking and 2-kb 3'-flanking DNA.
Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes.
Authors: Fisher R F , Egelhoff T T , Mulligan J T , Long S R
Published: June 2007

Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.
An 80-bp cis-acting regulatory region controls cAMP and development regulation of a prestalk gene in Dictyostelium.
Authors: Datta S , Firtel R A
Published: June 2007

We have analyzed an 80-bp region containing the cis-acting sequences necessary for regulation of the Dictyostelium discoideum prestalk gene pst-cathepsin at the appropriate stage during multicellular development and in response to cAMP in single-cell culture. The region lies between approximately -280 and -200 bp upstream from the Cap site and contains two intertwined G/C-rich sequences, including a palindromic sequence and a direct repeat of the 3' portion of the palindrome. In a previous set of experiments, we showed that the direct repeat, or G-box, is important in the regulation of pst-cath expression. In this paper, we use a series of nested internal deletions to define other regions within the promoter required for cAMP and developmental expression, to further examine the importance of the two G-boxes, and to examine the functional relationship of the G-boxes with respect to the other regulatory sequences. Analysis of the expression of these constructs in transformed cells showed that both the 5' portion of the palindrome and the two G-boxes are required for promoter function and are capable of developmentally regulating pst-cath expression. An A/T-rich sequence located 5' to the G/C-rich sequence is also essential for maximal expression, whereas insertion of a linker 5' to this region suggests the presence of a negative element functional during multicellular development. The spacing between the G-box sequences proved to be important for the full induction of gene expression. Constructs containing the G-boxes at wild-type spacing or closer show wild-type or near wild-type levels of expression, whereas expansion of the region between the G-boxes leads to a substantial drop in the level of gene expression in response to cAMP. Insertion of an oligonucleotide containing one of the G-boxes and surrounding sequences can partially complement deletions in which this region has been removed. Analysis of the expression of the cassette constructs, in some cases, revealed significant differences in the quantitative level of expression under the two developmental conditions. This suggests that there are either qualitative or quantitative differences in the factors controlling the expression of this gene under these two conditions.
A long, nontranslatable poly(A) RNA stored in the egg of the sea urchin Strongylocentrotus purpuratus.
Authors: Calzone F J , Lee J J , Le N , Britten R J , Davidson E H
Published: June 2007

Nontranslatable transcripts containing interspersed repetitive sequence elements constitute a major fraction of the poly(A) RNA stored in the cytoplasm of both the sea urchin egg and the amphibian oocyte. We report the first complete sequence of a representative interspersed maternal RNA transcript, called ISp1. The transcript is about 3.7 kb in length [including poly(A) tail]; and the 5' half consists of a cluster of repetitive sequences, whereas the 3' half is single copy. Other repetitive sequences occur in the 5' and 3' regions flanking the transcription unit. In several cloned alleles, the flanking repetitive and single-copy sequences differ, indicating a high degree of insertional and deletional rearrangement around, as well as within, the transcription unit. No significant open reading frames exist in any region of the ISp1 transcript, nor is it spliced to give rise to translatable mRNA in egg or embryo. A 620-nucleotide repetitive sequence element at the 5' end of the ISp1 transcript is also represented in a large number of other long interspersed maternal poly(A) RNAs. In addition, this sequence appears in a prevalent set of small polyadenylated RNAs about 600-nucleotides in length, which disappear almost completely by the gastrula stage of development. The structural features of the ISp1 RNA uncovered in this work exclude several hypotheses of interspersed maternal poly(A) RNA origin and function.
Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing.
Authors: Nelson K K , Green M R
Published: June 2007

To study the determinants of splice site selection, we have inserted synthetic 5' and 3' splice sites at different positions within beta-globin genes and analyzed the resultant RNA substrates for in vitro splicing, factor binding, and complex assembly. We show that consensus 5' and 3' splice site sequences are insufficient to determine splice site utilization; in the presence or absence of the authentic site, the synthetic sites are variably active in a position-dependent manner. However, regardless of position or utilization, the synthetic 5' and 3' splice sites are bound by the appropriate splicing factors. Thus, binding of splicing factors is necessary but not sufficient for splice site utilization. Finally, we demonstrate that a block to efficient splicing can occur at multiple steps in the pathway of normal splicing complex assembly.
Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons.
Authors: Miller S C , Pavlath G K , Blakely B T , Blau H M
Published: June 2007

Major changes in cytoarchitecture and gene expression were induced in short-term heterokaryons. When human hepatocytes were fused with mouse muscle cells, the hepatocyte Golgi apparatus changed from its usual polar location to a uniformly circumnuclear location typical of striated muscle. Human liver albumin ceased to be expressed, and expression of the human muscle cell-surface antigen 5.1H11 was induced without DNA replication or cell division. Coexpression of liver and muscle proteins was rarely observed. These novel findings provide insight into the regulation of gene expression and the targeting and localization of organelles with a central role in cell polarity, intracellular transport, and secretion.
Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain.
Authors: Davis C A , Noble-Topham S E , Rossant J , Joyner A L
Published: June 2007

We have examined the pattern of expression of the homeo box-containing gene En-2 during mouse embryogenesis using in situ hybridization. Transcripts were first detected in the neural folds of 8.0-day, 5-somite embryos, and expression continued throughout development into adulthood. Hybridization occurred only in the central nervous system (CNS) and was limited to one band of the neural tube and to parts of those structures that later developed from it; the cerebellum, pons, periaqueductal gray, and colliculi. Expression in the germinal zone of the CNS was uniform within the hybridizing band. However, later in development, once cells had migrated out of the germinal zone, there was a reduction in the extent of hybridization and an increase in its spatial complexity. In the adult, expression of En-2 appeared to be limited to specific groups of neurons. The early, localized expression of En-2 within an apparently homogeneous tissue is consistent with the hypothesis that En-2 plays a role in defining a spatial domain within the developing brain.