bluuebunny/crossref_metadata_2025 huggingface dataset:
Repression of ci-D in posterior compartments of Drosophila by engrailed.
Authors: Eaton S , Kornberg T B
Published: June 2007

A Drosophila strain was isolated that carries a lacZ-bearing recombinant P element integrated in the cubitus interruptus Dominant (ci-D) locus. This strain expresses beta-galactosidase in the anterior compartments of embryos and imaginal discs, suggesting that expression of ci-D is anterior-compartment specific. DNA from ci-D was cloned. In situ hybridization to imaginal discs confirmed that ci-D RNA is present only in anterior compartments. ci-D RNA is also limited to the anterior compartments at the germ-band extension stage of embryonic development, although not at earlier stages. In engrailed mutants, ci-D expression was derepressed in the posterior compartments of both embryos and imaginal discs. We postulate that engrailed, which is expressed in only the posterior compartments of embryos and imaginal discs, represses ci-D in these cells.
Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed.
Authors: Collier V L , Kronert W A , O'Donnell P T , Edwards K A , Bernstein S I
Published: June 2007

By comparing the structure of wild-type and mutant muscle myosin heavy chain (MHC) genes of Drosophila melanogaster, we have identified the defect in the homozygous-viable, flightless mutant Mhc10. The mutation is within the 3' splice acceptor of an alternative exon (exon 15a) that encodes the central region of the MHC hinge. The splice acceptor defect prevents the accumulation of mRNAs containing exon 15a, whereas transcripts with a divergent copy of this exon (exon 15b) are unaffected by the mutation. In situ hybridization and Northern blot analysis of wild-type organisms reveals that exon 15b is used in larval MHCs, whereas exons 15a and/or 15b are used in adult tissues. Because Mhc10 mutants fail to accumulate transcripts encoding MHC protein with hinge region a, analysis of their muscle-specific reduction in thick filament number serves as a sensitive assay system for determining the pattern of accumulation of MHCs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc10 adults reveals that those that contract rapidly or develop high levels of tension utilize only hinge region a, those that contract at moderate rates accumulate MHCs of both types, and those that are slowly contracting have MHCs with hinge region b. The presence of alternative hinge-coding exons and their highly tissue-specific usage suggests that this portion of the MHC molecule is important to the isoform-specific properties of MHC that lead to the different physiological and ultrastructural characteristics of various Drosophila muscle types. The absence of other alternative exons in the rod-coding region, aside from those shown previously to encode alternative carboxyl termini, demonstrates that the bulk of the myosin rod is not involved in the generation of isoform-specific properties of the MHC molecule.
Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.
Authors: Lasko P F , Ashburner M
Published: June 2007

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.
Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter.
Authors: Hama C , Ali Z , Kornberg T B
Published: June 2007

The Drosophila engrailed gene is expressed in the cells of the posterior developmental compartments. To investigate how the engrailed gene is regulated, chimeric genes consisting of fragments of the engrailed promoter and Escherichia coli lacZ were incorporated into the Drosophila germ line by P-element-mediated recombination. Fusion constructs with 7.5 kb of 5'-flanking sequence contain sufficient information to promote expression in most of the embryonic, larval, and imaginal posterior compartments; transformants with smaller fragments of the 5' region do not. Remarkably, of 20 independent transformants with constructs containing more than 1 kb of 5'-flanking DNA, 7 integrated in or around the engrailed locus. These strains inactivate engrailed function to varying degrees, and some express lacZ with a position- and temporal-specific program that is indistinguishable from the normal engrailed gene. Presumably, in these strains, lacZ is expressed in the context of the engrailed promoter.
Developmental and thyroid hormone-dependent regulation of pancreatic genes in Xenopus laevis.
Authors: Shi Y B , Brown D D
Published: June 2007

We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.
An RNA polymerase I termination site can stimulate the adjacent ribosomal gene promoter by two distinct mechanisms in Xenopus laevis.
Authors: McStay B , Reeder R H
Published: June 2007

On the ribosomal genes of Xenopus laevis, the T3 terminator is located approximately 60 bp upstream of the 5' boundary of the gene promoter. We have shown previously that mutation of the terminator simultaneously abolishes termination and impairs initiation by RNA polymerase I. Here, we show that the terminator influences the promoter by two distinct mechanisms. In one mechanism the terminator protects the promoter by preventing polymerase from reading through the initiation complex. In a second mechanism, the terminator interacts directly with the promoter, whether or not termination occurs. This positive interaction requires precise positioning of the terminator relative to the promoter and is sensitive to movement of the terminator by as little as 1 or 2 bp. We conclude that the terminator and promoter interact as one interdependent complex.
A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.
Authors: Roy S , Delling U , Chen C H , Rosen C A , Sonenberg N
Published: June 2007

The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions +23 to +25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position +23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions +31 to +34) and the stem (positions +9 to +12 and +49 to +52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.
Development-specific sigma-factor essential for late-stage differentiation of Myxococcus xanthus.
Authors: Apelian D , Inouye S
Published: June 2007

The gene for a developmentally expressed sigma-factor, sigB, has been isolated from Myxococcus xanthus by use of the sigA gene (formerly rpoD) of the vegetative sigma-factor as a probe. The sequence of sigB has been determined, and an open reading frame of 193 amino acid residues (Mr = 21,551) was identified. The amino-terminal region of SigB contains 69 residues, of which 35 are identical (50% identity) to the region of SigA required for core RNA polymerase binding and initiation of RNA polymerization. SigB also possesses many features commonly found in other prokaryotic sigma-factors. Analysis of an M. xanthus strain carrying a sigB-lacZ fusion gene revealed that sigB is expressed from a middle to late stage of differentiation corresponding to the period from the onset of sporulation to late development. A sigB deletion mutant displayed normal mound formation and sporulation; however, production of the ops gene product in myxospores of the delta sigB strain was shown to be blocked. Myxospores from the sigB deletion strain also exhibited severe defects in stability and viability during late development. Our data indicate that sigB encodes a sigma-factor essential for the maturation of myxospores at a late stage of M. xanthus differentiation. Our results also suggest that differentiation of M. xanthus is regulated by development-specific sigma-factors.
HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally.
Authors: Lai E , Prezioso V R , Smith E , Litvin O , Costa R H , Darnell J E
Published: June 2007

Hepatocyte-specific gene expression requires the interaction of many proteins with multiple binding sites in the regulatory regions. HNF-3 is a site found to be important in the maximal hepatocyte-specific expression of several genes. We find that liver nuclear extracts contain three major binding activities for this site, which we call HNF-3A, HNF-3B, and HNF-3C. Purification from rat liver nuclear extracts of HNF-3A and HNF-3C reveals that each activity corresponds to a distinct polypeptide, as determined by SDS-PAGE. Peptide sequence derived from the most abundant species, HNF-3A, was used for synthesizing probes with which to isolate a cDNA clone of this protein. The encoded protein contains 466 amino acids (48.7 kD) and has binding properties identical to those of the purified protein. A 160-amino-acid region that does not resemble the binding domain of any known transcription factor is essential for DNA binding. The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF-3A gene transcription.
ski can cause selective growth of skeletal muscle in transgenic mice.
Authors: Sutrave P , Kelly A M , Hughes S H
Published: June 2007

We have created several lines of mice that contain a truncated chicken c-ski cDNA linked to an MSV LTR promoter. Adult mice from three independent lines show large increases in skeletal muscle. All three lines of mice express high levels of c-ski mRNA and protein in skeletal muscle. All other tissues examined show little or no expression of the c-ski transgene. The muscles of one of the three lines were examined in more detail. Type II fast fibers undergo selective hypertrophy in affected muscles of this line.
In vitro polyadenylation is stimulated by the presence of an upstream intron.
Authors: Niwa M , Rose S D , Berget S M
Published: June 2007

The majority of vertebrate pre-mRNAs are both spliced and polyadenylated. To investigate the mechanism whereby processing factors recognize last exons containing both splicing and polyadenylation consensus elements, chimeric precursor RNAs containing a single intron and a poly(A) site were constructed and assayed for in vitro splicing and polyadenylation. Chimeric RNAs underwent splicing and polyadenylation. Both reactions occurred in a single RNA. The presence of an intron enhanced the rate of polyadenylation at a downstream poly(A) site. The extent of stimulation varied from two- to fivefold, depending on the magnesium concentration. Maximal stimulation of polyadenylation by an upstream intron required a 3' splice site but not a 5' splice site, suggesting that the structure of the terminal exon was more important than the presence of a complete upstream intron. We suggest that splicing and polyadenylation factors interact to recognize terminal, poly(A) site-containing exons. Such interaction may explain why all known intron-containing eukaryotic pre-mRNAs generate their 3' ends by polyadenylation.
A yeast protein that binds to vertebrate telomeres and conserved yeast telomeric junctions.
Authors: Liu Z P , Tye B K
Published: June 2007

We have identified three yeast proteins that bind to poly(C.A)/poly(T.G) repeats characteristic of telomeric sequences from yeast to human. TBF alpha binds to the telomeric sequences of yeast, Tetrahymena, and vertebrates. In contrast, TBF beta binds only to yeast telomeric sequences. Also identified was RAP1, the transcriptional silencer protein, which binds to a sequence motif found in upstream activating sequences (UASs) of a number of genes; the sequence motif also occurs frequently in yeast telomeric sequences. Because poly(C.A)/poly(T.G) sequences from a wide range of organisms will serve as the primer for the in vivo extension of telomeres in yeast, TBF alpha is of particular interest. DNase I footprinting analysis indicated that TBF alpha binds to the junction between the subtelomeric X sequence and poly(C1-3A) in a cloned yeast telomere. Examination of the junctions of known X sequences indicated that they all contain one or more repeats of CCCTAA, a sequence that is repeated in vertebrate telomeres. Earlier, Murray et al. (1988) reported that heterologous telomeric sequences positioned as far as several hundred base pairs from the termini of linear molecules can allow the addition of yeast telomeric sequences from nontelomeric termini in vivo. A possible function for TBF alpha might be to serve as an anchoring protein for the yeast telomerase by binding to the conserved junction sequence at a distance from the terminus to allow addition of an irregular repeating sequence at the chromosome end.
DNA-protein interaction at the replication termini of plasmid R6K.
Authors: Sista P R , Hutchinson C A , Bastia D
Published: June 2007

Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific DNA sequences (tau), constituting the replication terminus. Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites. With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis. The results show that one monomer of ter protein binds to a single tau site with an equilibrium dissociation constant of 5 x 10(-9) moles/liter. Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the tau core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo. The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a tau sequence. Thus, the asymmetric ter-tau complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a tau site.
cAMP and cell sorting control the spatial expression of a developmentally essential cell-type-specific ras gene in Dictyostelium.
Authors: Esch R K , Firtel R A
Published: June 2007

The Dictyostelium ras gene (Dd-ras) is expressed at a low level in vegetative cells, is not expressed between the onset of development and aggregation, and is then re-expressed in the multicellular aggregate stages from the distal, now cAMP-responsive, promoter and from two more proximal promoters. Expression of activated Dd-ras (G12----T12) (Reymond et al. 1986) results in an abnormal developmental phenotype with the formation of aggregates having multiple tips and an inhibition of further development. In this report we investigate the spatial expression of Dd-ras by fusing the 5'-flanking region to the Escherichia coli lacZ gene and by staining aggregates for beta-galactosidase (beta-gal) activity. We show that fusions using 5'-flanking sequences that include all promoters are expressed in approximately 10-20% of the cells randomly scattered within the early aggregate. Our data indicate that these beta-gal-expressing cells migrate to newly formed tips of aggregates and localize in the region that becomes the prestalk zone. Staining is also seen in the very posterior of the organism. The anterior staining appears to be specific for the prestalk A population, and beta-gal activity is subsequently present in stalk cells as developmental proceeds. When only the two more proximal promoters are used to drive lacZ expression, localized staining is seen in the anterior prestalk region, although it is weaker than with the construct carrying all promoters. Moreover, staining is not seen in the posterior domain in the first finger stage, suggesting differences in the spatial expression from the different promoters. Staining is also observed in some cells within the prespore region, which could be anterior-like cells. The pattern of Dd-ras/lacZ staining during tip formation suggests a directed, spiral pattern of cell migration, possibly in response to the proposed spiral gradient of cAMP within the developing aggregate. The pattern of Dd-ras is consistent with the abnormal developmental phenotype caused by expressing an activated Dd-ras Thr12 gene and suggests an essential role for Dd-ras in controlling spatial differentiation.
HOX-4 genes and the morphogenesis of mammalian genitalia.
Authors: Dollé P , Izpisúa-Belmonte J C , Brown J M , Tickle C , Duboule D
Published: June 2007

We examined the temporal and spatial expression patterns of the homeo box HOX-4 complex genes during the morphogenesis of the genitalia of mice. The results show that only those Hox-4 genes that are expressed very posteriorly in the trunk, or very distally in the limbs, seem to be involved in the patterning of the genital tubercle. This is consistent with the idea of "temporal colinearity", which suggests that the very last structure to require patterning during vertebrate development will express Hox genes located at the 5' extremity of the HOX complexes. We also show that genital tubercle mesenchyme can respecify pattern in the chicken wing bud. This finding reinforces the concept of the uniformity in the patterning mechanisms along the various axes of the body.
Temporal regulation of lin-14 by the antagonistic action of two other heterochronic genes, lin-4 and lin-28.
Authors: Arasu P , Wightman B , Ruvkun G
Published: June 2007

Heterochronic genes form a regulatory pathway that controls the temporal sequence of the Caenorhabditis elegans postembryonic cell lineage. One of these genes, lin-14, encodes a nuclear protein that constitutes a temporal developmental switch. During wild-type development, lin-14 protein is abundant during early larval stage 1 (L1) to specific L1-specific cell lineages but is nearly undetectable at L2 and later stages to specify L2-specific and later cell lineages. To determine the roles played by other genes in executing this temporal switch, we have analyzed how lin-14 expression is regulated by other heterochronic genes. lin-4 is required to down-regulate lin-14 protein levels during the L1 stage, whereas lin-28 positively regulates lin-14 protein levels. The lin-4 gene product is a candidate for interacting with the negative regulatory element in the 3'-untranslated region of lin-14. lin-29 mutations do not affect lin-14 protein levels, consistent with lin-29 acting downstream of lin-14. Switching off lin-14 expression during the L1 stage is not triggered by the passage of time per se but, rather, is normally dependent on feeding or the feeding-dependent initiation of postembryonic cell division.
Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.
Authors: Gattoni R , Chebli K , Himmelspach M , Stévenin J
Published: June 2007

The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAs) by the use of three alternative 5'-splice sites. The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively. With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells. An in vitro analysis of the cis-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction. The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides. Complementation experiments show that the trans-acting factor(s) are micrococcal nuclease sensitive. They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection. We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells.
The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase.
Authors: Nodwell J R , Greenblatt J
Published: June 2007

The boxA and boxB components of the lambda nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, S10, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA.
v-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes.
Authors: Metz T , Graf T
Published: June 2007

E26 is an acute avian leukemia virus that encodes the transcriptional activator oncogenes v-myb and v-ets in a single fusion protein. This virus is also unique in that it is able to transform hematopoietic cells of both the myeloid and the erythroid lineage. To determine the contributions of v-myb and v-ets to the transforming potential of the virus, derivatives expressing separate Myb and Ets proteins, either alone or in combination, were constructed. We found that in the myeloid lineage v-myb, but not v-ets, induces cell transformation. In the erythroid lineage both v-myb and v-ets weakly transform erythroblast-like cells. These cells exhibit a mature phenotype and a low self-renewal capacity. The transforming efficiency of the two oncogenes is enhanced if they are coexpressed as separate proteins or as a fusion protein, the transformed cells displaying an increased self-renewal capacity. Interestingly, however, cells transformed by the Myb-Ets fusion protein have a distinct phenotype in that they are very immature. These results demonstrate that v-myb and v-ets can cooperate in the transformation of erythroid cells both in trans and in cis and that the mode of cooperation is reflected by the differentiation phenotypes of the transformed cells.
Developmental distribution of female-specific Sex-lethal proteins in Drosophila melanogaster.
Authors: Bopp D , Bell L R , Cline T W , Schedl P
Published: June 2007

The binary switch gene Sex-lethal (Sxl) must be on in females and off in males to allow the proper elaboration of the appropriate sexual developmental pathway in Drosophila melanogaster. Previous studies suggested a mechanism in which the on/off regulation of Sxl occurs post-transcriptionally at the level of RNA splicing. A critical prediction of this model is that functional Sxl proteins are absent in males but present in females. In this report we show that the expected full-length proteins are only present in female animals. Multiple forms of Sxl protein are found in females, some of which are expressed in a stage- and tissue-specific pattern. Consistent with a role of Sxl proteins in regulating alternate splicing, the proteins are localized in the nucleus where they exhibit a punctate staining pattern. Surprisingly, several minor Sxl proteins appear to be present in specific tissues of both sexes of adults. The possible origin of these species is discussed. We also show that Sxl expression in the early embryo is sex specific and depends on maternal daughterless and zygotic sisterless-b activity in accordance with the established roles of these genes as positive regulators of Sxl. The onset of Sxl expression in the germ line occurs later than that in the soma.
Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.
Authors: Twell D , Yamaguchi J , Wing R A , Ushiba J , McCormick S
Published: June 2007

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.
A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1).
Authors: Shero J H , Hieter P
Published: June 2007

A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed. A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation. This method identified two suppressors, designated MCK1 and CMS2. Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at 11 degrees C), and grew slowly on Benomyl (a microtubule-destabilizing drug). Furthermore, when grown at 18 degrees C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss. The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function. The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation. MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity.
Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing.
Authors: Izban M G , Luse D S
Published: June 2007

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase II transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCl. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.
The phage T4 nrdB intron: a deletion mutant of a version found in the wild.
Authors: Eddy S R , Gold L
Published: June 2007

Bacteriophage T4 possesses three self-splicing group I introns. Two of the three introns are mobile elements; the third, in the gene encoding a subunit of the phage nucleotide reductase (nrdB), is not mobile. Because intron mobility offers a reasonable explanation for the paradoxical occurrence of large intervening sequences in a space-efficient eubacterial phage, it is puzzling that the nrdB intron is not mobile like its compatriots. We have discovered a larger nrdB intron in a closely related phage, and we infer from comparative sequence data that the T4 intron is a deletion mutant derived from this larger intron. This larger nrdB intron encodes an open reading frame of 269 codons, which we have cloned and overexpressed. The overexpressed protein shows a dsDNA endonuclease activity specific for the intronless nrdB gene, typical of mobile introns. Thus, we believe that all three introns of T4 are or were mobile "infectious introns" and that they have entered into and been maintained in the phage population by virtue of this efficient mobility.
HNF-1 alpha and HNF-1 beta (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro.
Authors: Mendel D B , Hansen L P , Graves M K , Conley P B , Crabtree G R
Published: June 2007

HNF-1 alpha (previously referred to as HNF-1, LPB1, and APF) is a vertebrate transcription factor that contains a divergent homeo domain and plays a prominent role in regulating genes that have the common characteristic of being expressed in hepatocytes and a complex group of endodermally and mesodermally derived tissues. HNF-1 alpha is unique among the vertebrate homeo domain-containing proteins in that it dimerizes in the absence of its DNA recognition sequence, suggesting the possibility that the function of HNF-1 alpha may be diversified by forming heterodimers with other related proteins. We report the initial characterization of HNF-1 beta, which is closely related to HNF-1 alpha and is able to form heterodimers with HNF-1 alpha in vitro. Although HNF-1 alpha, but not HNF-1 beta, is expressed in the liver, HNF-1 alpha and HNF-1 beta are coexpressed in the murine Hepa1A cell line and in the mammalian kidney where a subset of hepatocyte genes are expressed. In contrast, exclusive expression of HNF-1 beta is associated with repression of a subset of hepatocyte-specific genes in the dedifferentiated hepatocyte cell line C2, differentiated F9 cells, in somatic hybrids between hepatocytes and fibroblasts, and in the lung. The extent of heterodimerization may be regulated in a tissue-specific way because freely exchangeable heterodimers are formed in Jurkat T cells transfected with HNF-1 alpha and HNF-1 beta, whereas in liver cells stable homodimers are present. These studies define a pair of homeo domain proteins that have the potential to interact to produce an embryologically complex pattern of gene expression.
Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.
Authors: Ben-David Y , Giddens E B , Letwin K , Bernstein A
Published: June 2007

The retroviral integration site Fli-1 is rearranged in 75% of the erythroleukemia cell clones induced by Friend murine leukemia virus (F-MuLV), whereas Spi-1/PU.1, a member of the ets family of DNA-binding proteins, is rearranged in 95% of the erythroleukemias induced by Friend spleen focus-forming virus (SFFV). To determine the transcriptional domain defined by Fli-1, we have isolated a cDNA clone that is highly expressed only in erythroleukemia cell lines with Fli-1 rearrangements. The protein sequence of this cDNA is very similar to Erg2, another member of the ets gene family. The hydrophilic carboxy-terminal end of the Fli-1 cDNA shares significant sequence similarity to the DNA-binding ETS domain found in all members of the ets family. PFGE analysis localized Fli-1 within 240 kb of the ets-1 proto-oncogene on mouse chromosome 9 and human chromosome 11q23, suggesting that ets-1 and Fli-1 arose from a common ancestral gene by gene duplication. The involvement of the murine Fli-1, Spi-1, and avian v-ets genes in erythroleukemia induction suggests that activation of ets gene family members plays an important role in the progression of these multistage malignancies.
Cell cycle-specific expression of the SWI4 transcription factor is required for the cell cycle regulation of HO transcription.
Authors: Breeden L , Mikesell G E
Published: June 2007

Expression of the HO endonuclease triggers mating-type switching in Saccharomyces cerevisiae. Transcription of the HO gene is start-dependent and restricted to the late G1/early S phase of haploid mother cells. The HO promoter contains 10 copies of a cell cycle-regulated upstream activation sequence, which is activated by SWI4 and SWI6. SWI4 mRNA levels vary at least 10-fold throughout the cell cycle and rise sharply just before the rise in HO mRNA levels. Constitutive synthesis of SWI4 mRNA leads to constitutive synthesis of HO mRNA. These data suggest that the cell cycle regulation of SWI4 mRNA is required for the tight cell cycle regulation of HO transcription. High-level constitutive synthesis of SWI4 also suppresses swi5 and swi6 mutations, suggesting that SWI4 is the predominant activator of HO transcription and that mutations in negative regulators of SWI4 could be isolated as suppressors of swi6 mutations. One recessive suppressor of swi6 (ssx1-1) that allowed high-level expression of SWI4 during alpha-factor arrest and constitutive expression of both SWI4 and HO after release from the arrest was isolated. This result suggests that SSX1 has a negative regulatory role in the cell-cycle regulation of SWI4 mRNA accumulation.
Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron.
Authors: Deshler J O , Rossi J J
Published: June 2007

The 3' splice site of the budding yeast Kluyveromyces lactis actin gene (ACT) intron is distally spaced (122 nucleotides) from its branchpoint and is also preceded by a silent PyAG located 43 nucleotides upstream. We devised a genetic screen that resulted in the isolation of several randomly induced cis-acting mutations that activate the silent PyAG as a 3' splice site. These mutations fall within a region surrounding this PyAG, which can hypothetically fold into a higher-order structure. Site-directed mutational analyses demonstrate that a hairpin structure in this region is required for correct 3' splice-site selection. Analysis of the point mutations suggests that local breathing of the hairpin near the first PyAG can lead to its activation. These data demonstrate that 3' splice-site selection is not a consequence of a linear, directional scanning mechanism, but support the notion of a critical positioning requirement for 3' splice-site selection. We speculate on the possible origin of this intron-encoded structural motif, which has homology to a bacterial transposon and suggests one possible origin for alternative splicing mechanisms in higher eukaryotes.
Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains.
Authors: Taylor I C , Workman J L , Schuetz T J , Kingston R E
Published: June 2007

Regulatory factors must contend with chromatin structure to function. Although nucleosome structure and position on promoters can be important in determining factor access, the intrinsic ability of factors to bind to nucleosomal DNA might also play an essential regulatory role. We have used templates where nucleosomes were either randomly positioned or rotationally phased to demonstrate that two transcription factors, heat shock factor (HSF) and GAL4, differ significantly in their ability to bind to nucleosomes. GAL4 was able to bind to nucleosomal templates. Surprisingly, in contrast to its behavior on naked DNA, GAL4 bound better to multiple GAL4 sites than to a single GAL4 site on these templates. HSF alone was not able to bind to nucleosomal templates. HSF was able to bind to nucleosomal templates, however, when the TATA-binding factor TFIID was present. Consequently, binding to nucleosomal templates could be facilitated by adjacent binding of the same protein in the case of GAL4 but required binding of a second protein in the case of HSF. Taken together, these data demonstrate that regulatory factors differ in their inherent ability to bind to nucleosomal templates. These differences are likely to be important to the function of these factors in vivo.
Transcriptional regulation of a yeast HSP70 gene by heat shock factor and an upstream repression site-binding factor.
Authors: Park H O , Craig E A
Published: June 2007

SSA1, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, displays a significant basal level of expression under optimal growth conditions. Although multiple sites related to the heat shock element (HSE) consensus sequences are present in the SSA1 promoter region, one of these, HSE2, plays a key role in basal expression. An upstream repression site (URS) located adjacent to HSE2 causes repression of basal expression but has little effect on heat-inducible expression of SSA1. A series of DNase I footprinting assays suggests that heat shock factor (HSF) and a URS-binding factor (URSF) can bind simultaneously to the adjacent binding sites HSE2 and URS under optimal growth conditions. URSF in extracts from heat-shocked cells does not bind (or binds very poorly) to the URS adjacent to HSE2. However, URSF in these extracts is able to bind the URS if the URS is separated from HSE2 or if the HSE is mutated such that HSF binding is abolished. These in vitro experiments are consistent with in vivo results showing that the URS is able to repress transcription driven by HSE2 both before and after heat shock if it is separated from HSE2. Our results are consistent with a model of repression in which URSF and HSF bind simultaneously to the adjacent binding sites URS and HSE2 prior to heat shock. After heat shock, however, binding of the two proteins to the adjacent sites is exclusive, perhaps due to modification of HSF known to occur upon heat shock. Because HSF binding predominates, repression by URSF is relieved upon heat shock.
Retinoic acid perturbs the expression of Xhox.lab genes and alters mesodermal determination in Xenopus laevis.
Authors: Sive H L , Cheng P F
Published: June 2007

Retinoic acid (RA) treatment of Xenopus laevis embryos leads to anterior truncation of the body axis (Durston et al. 1989; Sive et al. 1990). These initial studies suggested that RA may play a role in the patterning of the primary body axis. At least one target of RA was shown previously to be dorsal ectoderm. In this report we extend this observation and also ask whether RA alters the determination and inducing capacity of mesodermal tissue. To facilitate this analysis we isolated the homeo-domain-containing genes Xhox.lab1 and Xhox.lab2. These genes were expressed in both ectoderm and mesoderm during the RA-sensitive period and were strongly induced by RA in both germ layers. In particular, anterior regions expressed low levels of Xhox.lab RNAs in untreated embryos but showed increased expression after RA application. We show further that although RA-treated embryos contained anterior-inducing mesoderm, the amount of this activity appeared to be lower than that of controls. Additionally, we document that RA suppressed lateral (muscle) and ventral (blood) mesoderm differentiation. The data indicate that RA alters mesodermal determination and causes axial perturbation both by depressing the ability of dorsal mesoderm to induce anterior structures and by altering the response of dorsal ectoderm to induction. These analyses suggest that Xhox.lab genes may be responsible, in part, for mediating the RA effect.
The pituitary-specific regulatory gene GHF1 contains a minimal cell type-specific promoter centered around its TATA box.
Authors: McCormick A , Brady H , Fukushima J , Karin M
Published: June 2007

GHF-1 is a pituitary-specific transcription factor responsible for activation of the growth hormone (GH) gene. The GHF1 gene is expressed exclusively in cells of the somatotrophic lineage, and its transcription is extinguished in somatic cell hybrids. The minimal sequences required for differential transcription of GHF1 in GH-expressing and -nonexpressing cell lines and somatic cell hybrids were localized to a 15-bp region surrounding and including its TATA box. This 15-bp fragment acts as a cell type-specific promoter element and is recognized by a transcription factor present in GH-expressing cell lines. Hence, in addition to enhancers and upstream promoter elements, the TATA element (TATA box plus surrounding sequences) can be, in certain cases, an important determinant of cell-type-specific transcription.
Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10.
Authors: Mason S W , Greenblatt J
Published: June 2007

The transcription antitermination protein, N, of bacteriophage lambda; the Escherichia coli elongation factors NusA, NusB, ribosomal protein S10, and NusG; and a DNA template containing a lambda nut (N-ututilization) site are necessary and sufficient for the highly cooperative formation in vitro of stable transcription complexes containing all five elongation factors. Mutations in the nut site, NusA, or the beta-subunit of RNA polymerase (RNAP) that impair antitermination in vivo also abolish the assembly of a stable complex containing the antitermination factors in vitro. The effects of RNAP mutations on assembly imply that the antitermination factors assemble on the surface of RNAP. We have shown previously that NusA binds directly to transcribing RNAP (Ka approximately 10(7) M-1); Ka = association constant and we show here that S10 also binds directly and specifically to RNAP with an apparent Ka of 10(6) M-1. These observations led to a model for the ordered assembly of the N-modified transcription complex.
The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex.
Authors: Dostatni N , Lambert P F , Sousa R , Ham J , Howley P M , Yaniv M
Published: June 2007

The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.