bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic rearrangements and gene amplification in Escherichia coli: DNA sequences at the junctures of amplified gene fusions.
Authors: Whoriskey S K , Nghiem V H , Leong P M , Masson J M , Miller J H
Published: June 2007

We describe gene fusions that result from genetic duplications of 5-20 kb, which are amplified 50- to 100-fold. Because one end point of the fusion lies within the sequenced lacI gene, the new junctures created by the duplications are readily identified. Using a procedure for dideoxy sequencing of double-stranded DNA, we show that the duplications occur almost exclusively at short sequence repeats (less than 15 bp), sometimes involving broken homologies, in the 30 cases examined. Most of the duplications place the lacI-Z encoded hybrid repressor-beta-galactosidase protein under the control of a downstream promoter, resulting in the production of a more complex hybrid protein with beta-galactosidase activity. In some cases the fusion occurs with the lacY or the lacA gene, which suggests that silent promoters can be uncovered by gene fusion and subsequent amplification. In some ways this system represents a bacterial analog to chromosomal rearrangements of oncogenes in higher cells, since here the expression of a silent gene is the result of a genetic rearrangement that is followed by amplification during selected growth.
Novel transcripts from the Ultrabithorax domain of the bithorax complex.
Authors: Lipshitz H D , Peattie D A , Hogness D S
Published: June 2007

We present a detailed analysis of the transcriptional products of the bithoraxoid (bxd) region of the Ultrabithorax domain in the bithorax complex of Drosophila melanogaster. This region is transcribed twice during development: between 3 and 6 hr of embryogenesis, a set of early transcripts, 1.1 to 1.3 kb in size, is synthesized; from the midthird larval instar through the adult stages, a late 0.8-kb transcript is synthesized. We have sequenced five cloned cDNAs representing early transcripts and three cDNAs representing the late transcript and have located their exons within the 40 kb of DNA comprising the bxd region. S1 nuclease protection and primer extension of both the early and late transcripts were used to further elucidate their structure. The early RNAs are produced by complex differential splicing of a series of exons derived from a 26-kb primary transcript. Curiously, these RNAs do not possess significant protein coding potential. The late bxd RNA comprises a single exon transcribed from an intronic region of the early transcription unit. This RNA, by contrast, possesses excellent coding potential and, if translated, would yield a 101-amino-acid polypeptide.
Nucleic acid splicing events occur frequently during macronuclear development in the protozoan Oxytricha nova and involve the elimination of unique DNA.
Authors: Ribas-Aparicio R M , Sparkowski J J , Proulx A E , Mitchell J D , Klobutcher L A
Published: June 2007

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus which contains exclusively linear, gene-sized DNA molecules with an average size of about 2.2 kilobase pairs (kbp). The micronuclear precursors of two macronuclear DNA molecules have been examined. Each was found to contain at least five blocks of DNA sequences that are absent in the mature macronuclear DNA molecule. These blocks of sequences, referred to as internal eliminated sequences (IESs), must be removed by a nucleic acid breakage and joining process during development. The data obtained to date indicate that IESs are common and suggest that greater than 60,000 IES removal events occur during macronuclear development. Additional analyses indicate that IESs represent a portion of the unique micronuclear DNA sequences known to be eliminated during development. Comparisons of the sequences of IESs revealed common organizational features and some limited primary sequence homologies that suggest models for their developmental excision.
Post-transcriptional restriction of gene expression in sea urchin interspecies hybrid embryos.
Authors: Conlon R A , Tufaro F , Brandhorst B P
Published: June 2007

The synthesis of many paternal species-specific proteins is reduced in all stages of sea urchin interspecies hybrid embryos, due to the reduced amounts of some paternal mRNA species in hybrid embryos compared with the embryos of the paternal species (Tufaro and Brandhorst 1982). Possible explanations for this restriction were tested. Cloned cDNAs were selected that were specific for paternal RNA sequences having reduced amounts (to 2-20% of normal) in hybrid embryos derived from a cross of Stronglyocentrotus purpuratus eggs with Lytechinus pictus sperm. Several of these RNA species are barely detectable in the eggs, but they accumulate extensively (5- to 40-fold) during L. pictus embryogenesis. Thus, the restricted expression of these paternal genes in hybrid embryos is not the result of the persistence of stable maternal mRNA species stored in eggs and not replaced by zygotic transcription. The accumulation of some of these L. pictus transcripts is also reduced in the reciprocal cross (L. pictus eggs X S. purpuratus sperm); therefore, the full expression of these L. pictus genes in hybrid embryos is not dependent on species-specific maternal factors stored in the egg. The transcriptional activity of one such gene was estimated using a run-on assay in isolated nuclei; it is as actively transcribed in hybrid as it is in homospecific embryos, but in hybrid embryos the cytoplasmic transcript accumulates to only 2-15% of the normal level. Sequence analysis indicates that this gene encodes a metallothionein. Mechanisms are discussed that might account for the post-transcriptional restriction of expression of some genes in hybrid embryos.
Activation and repression of mammalian gene expression by the c-myc protein.
Authors: Kaddurah-Daouk R , Greene J M , Baldwin A S , Kingston R E
Published: June 2007

One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the level of appropriately initiated expression from the human heat shock protein 70 (hsp70) promoter. Sequences required for full activation lie upstream of the transcription initiation site and are distinct from sequences necessary for basal expression. These sequences also appear distinct from promoter sequences necessary for heat induction, serum induction, and induction by the papovavirus T antigens. The c-myc protein inhibits appropriately initiated expression from the mouse metallothionein I (MT-I) promoter. A mutation that removes 138 amino acids of exon 2 produces a c-myc gene product that is capable of activating the hsp70 promoter but is no longer capable of inhibiting MT-I expression, suggesting that these two properties reside in different domains of the c-myc protein. Expression from the adenovirus EII promoter is slightly inhibited, while expression from the SV40 early promoter is minimally affected by the c-myc protein. Both the spectrum of promoters regulated by the c-myc protein and the sequence requirements for that regulation differ from those of previously characterized viral trans-activating proteins. The data suggest that the c-myc protein can both stimulate and inhibit transcription from mammalian promoters in a novel manner.
Retroviral transfer and expression of the interleukin-3 gene in hemopoietic cells.
Authors: Wong P M , Chung S W , Nienhuis A W
Published: June 2007

A recombinant retrovirus containing the interleukin-3 (IL3) coding sequence and the neomycin-resistance gene (Neor) has been generated. Infection of fetal liver cells with the IL3 retrovirus, but not with the N2 parental virus, resulted in the formation of factor-independent, NeoR colonies containing various types of differentiated hemopoietic cells. Established cell lines could be generated from these mixed hemopoietic colonies. These cell lines contained the unrearranged viral genome, produced viral IL3, and secreted the growth factor; however, they were not tumorigenic. Identical results were obtained from infection of two factor-dependent cell lines with the IL3 virus, except that these clones all became tumorigenic. These data indicate that endogenous IL3 production can support normal differentiation and immortalization of primary hemopoietic cells, or, in previously immortalized cells, can lead to tumorigenicity.
Human neoplastic B cells express more than two isotypes of immunoglobulins without deletion of heavy-chain constant-region genes.
Authors: Kinashi T , Godal T , Noma Y , Ling N R , Yaoita Y , Honjo T
Published: June 2007

Flow cytometric analyses of surface immunoglobulins of human neoplastic B cells from four patients indicated that more than two isotypes were expressed on the surface of each patient's neoplastic B cells. Southern blot analysis of DNAs of these cells showed that each patient's neoplastic cells had the monoclonal rearrangement profile of the J segment of the immunoglobulin heavy-chain gene. Heavy-chain constant-region genes of these cells had no deletion associated with S-S recombination. Northern blot analysis of RNA from two neoplastic cells revealed that each RNA contained comparable amounts of mRNAs for two different isotypes which were identified by surface staining. These results support the hypothesis that the simultaneous expression of two different isotypes in a certain stage of B-cell differentiation is mediated by alternative RNA splicing without DNA deletion.
Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli.
Authors: VanBogelen R A , Acton M A , Neidhardt F C
Published: June 2007

The addition of isopropyl thio-beta-D-galactoside (IPTG) to Escherichia coli cells containing multiple copies of the heat shock regulatory gene htpR (rpoH) under the control of an IPTG-inducible promoter (P-tac) induced 15 of the 17 polypeptides of the heat shock (HTP) regulon. The time course and magnitude of the induction closely resembled that caused by a shift to 42 degrees C. Nevertheless the two means of inducing the heat shock regulon differed in outcome. Cultures grown at 28 degrees C and induced by incubation at 42 degrees C for 15 min gave significant protection against a challenge temperature of 50 degrees C, but no protection was afforded by a 15-min IPTG treatment at 28 degrees C. It could be shown that there was no interference by IPTG with the development of thermotolerance at 42 degrees C. Also, treatment of a wild strain of E. coli with various toxic agents revealed no correlation between the development of thermotolerance and the induction of any subset of the heat shock proteins. Thermotolerance appears to develop by processes other than the htpR-dependent induction of heat shock proteins.
Function and misfunction of the two promoters of the Drosophila Antennapedia gene.
Authors: Jorgensen E M , Garber R L
Published: June 2007

In the Antennapedia (Antp) gene of Drosophila melanogaster, structurally distinct RNAs arise from different transcription initiation sites. When the two sites are separated by a chromosome inversion, transcripts are produced from each fragment of the split Antp locus, and these RNAs initiate at the same nucleotide as in wild-type animals. Thus, the initiation sites are regulated by independent promoters. We show by in situ hybridization that transcripts from each promoter accumulate in spatially distinct patterns in a subset of wild-type imaginal discs. Importantly, these patterns are generally maintained in the inversion mutant. We conclude that the promoters possess independent and dissimilar regulatory elements for spatial activation. Finally, we have looked at transcription in seven different dominant Antp mutants, all of which show a transformation of head tissue to thoracic tissue. In each mutant, the second promoter is improperly activated in the eye-antennal imaginal disc. Because all but one of these mutations have inversion breakpoints distantly upstream of the activated promoter, they probably act via long-range euchromatic position effects. Our studies define how the dual promoters and chromatin structure of the Antp gene contribute to the generation of a complex pattern of transcription.
Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators.
Authors: Kumamoto A A , Miller W G , Gunsalus R P
Published: June 2007

DNase I footprinting and methylation protection studies have been used to analyze the binding of Escherichia coli Trp repressor to the trpR, aroH, and trp operators. The methylation protection assay shows that Trp repressor binds in two successive major grooves of the trpR operator, three successive major grooves of the aroH operator, and four successive major grooves of the trp operator. The simplest model that explains the difference in Trp repressor interaction at the three operators is that the aroH and trp operators are composed of multiple, helically stacked binding sites. When viewed in three dimensions, each site is positioned on a different face of the DNA, and together process up the surface of the DNA helix. Analysis of a deletion derivative of the trp operator supports this model.
Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation.
Authors: Ohtsubo M , Kai R , Furuno N , Sekiguchi T , Sekiguchi M , Hayashida H , Kuma K , Miyata T , Fukushige S , Murotsu T
Published: June 2007

The human RCC1 gene was cloned after DNA-mediated gene transfer into the tsBN2 cell line, which shows premature chromosome condensation at nonpermissive temperatures (39.5-40 degrees C). This gene codes for a 2.5-kb poly(A)+ RNA that is well conserved in hamsters and humans. We isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone. The base sequences of these two active cDNA clones differ at the 5' proximal end, yet both have a common open reading frame, encoding a protein of 421 amino acids with a calculated molecular weight of 44,847 and with seven homologous repeated domains of about 60 amino acids. This human RCC1 gene was located to human chromosome 1 using sorted chromosomal fractions.
Identification in transgenic animals of the Drosophila decapentaplegic sequences required for embryonic dorsal pattern formation.
Authors: Hoffmann F M , Goodman W
Published: June 2007

Mutant alleles of the Drosophila decapentaplegic (dpp) gene affect embryonic dorsal-ventral pattern formation, larval viability, and adult cuticle formation from the imaginal disks. The dpp DNA required for this array of functions spans almost 50 kb. We report that the embryonic lethal, ventralizing alleles of the dpp gene are rescued in transgenic animals by an 8-kb fragment of the wild-type dpp DNA. Full rescue, from embryonic lethality to adult viability, is obtained in two situations: in animals hemizygous for the haplolethal dpp gene, and in animals hemizygous for either of two recessive embryonic lethal alleles. In embryos null for dpp, the transformation of dorsal cuticle to ventral cuticle is blocked by one copy of the dpp transposon; two copies permit the hatching of the larvae. The portion of dpp sufficient for these embryonic functions encodes a protein with homology to the transforming growth factor-beta (TGF-beta) family of proteins (Padgett et al. 1987). The larval and imaginal disk functions of dpp are not rescued by the 8-kb portion of the gene and must require additional sequences from the 50 kb of DNA.
Regulatory elements that modulate expression of human c-myc.
Authors: Hay N , Bishop J M , Levens D
Published: June 2007

Regulation of transcription from the proto-oncogene c-myc apparently plays an important part in cellular proliferation and the genesis of diverse tumors. Here, we report that the abundance of transcripts from the two principal promoters for human c-myc (P1 and P2) is governed by a composite of positive and negative regulators, located within a 2.3-kb domain upstream of the gene. In actively proliferating cells, the action of the positive elements is apparently dominant over that of the single negative regulator that we have identified. Nuclear proteins bind specifically to nucleotide sequences within the negative regulator and at least one of the positive regulators. The cooperative and counteracting actions of the regulatory elements described here presumably contribute to the plasticity of transcription from c-myc and may be affected by the tumorigenic damage that sometimes afflicts c-myc.
Electrophoretic separation of polyadenylation-specific complexes.
Authors: Skolnik-David H , Moore C L , Sharp P A
Published: June 2007

A polyadenylation-specific complex composed of precursor RNA containing the adenovirus-2 L3 site and HeLa cellular components was detected by electrophoresis on a native, low-percentage polyacrylamide gel. Upon incubation in a reaction containing ATP and nuclear extract, precursor RNA was rapidly assembled into this complex. This assembly did not require poly(A) synthesis, as it occurred efficiently in the presence of ATP analogs that inhibited this reaction. Mutation of the hexanucleotide AAUAAA 20 nucleotides upstream of the L3 site to AAGAAA or deletion of sequence between +5 and +48 nucleotides downstream of the L3 site inactivates polyadenylation. The specific complex did not effectively from on substrate RNA with either the AAGAAA mutation or the downstream deletion mutation. Kinetic experiments showed that the assembly of this complex preceded processing of precursor RNA. We proposed that formation of this complex represents an intermediate step in polyadenylation.
Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis.
Authors: Zeller R , Bloch K D , Williams B S , Arceci R J , Seidman C E
Published: June 2007

Expression of the gene encoding atrial natriuretic factor (ANF) during mouse cardiac embryogenesis was studied using in situ hybridization to histologic sections. ANF mRNA was first detected in a subpopulation of myocardial cells at day 8 of embryogenesis. During day 9, abundant hybridization of the ANF probe to the atrium and the primitive ventricle was found. At day 14, strong labeling of cells in both atria and the trabeculated regions of the left ventricle was seen, whereas fewer cells in the right ventricle expressed the ANF gene. The developmental regulation of ANF gene expression in atrial and ventricular cells suggests an important role for this peptide hormone in the embryo. Localized expression of the ANF gene provides a marker for discerning subpopulations of cardiac cells during embryogenesis. Identification of embryonic ventricular ANF mRNA transcripts may have implications with respect to pathologic reexpression of the ANF gene in adult ventricles.
A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans.
Authors: Hodgkin J
Published: June 2007

The normal sexes of Caenorhabditis elegans are the self-fertilizing hermaphrodite (XX) and the male (XO). The autosomal gene tra-1 is a major switch gene controlling sexual phenotype. Mutant phenotypes of 43 loss-of-function (lf) tra-1 alleles and 22 gain-of-function (gf) tra-1 alleles are described and discussed. The tra-1(lf) alleles are recessive and, in general, masculinizing. The most severe mutations (such as seven out of eight identified amber alleles) can transform XX animals into fertile males. These mutations have little effect on XO animals (which are male already) but lead to some abnormalities in XO gonadal development, indicating that tra-1 has functions in normal development of both sexes, although its major function is confined to the XX hermaphrodite. Weaker tra-1(lf) alleles lead to incomplete masculinization of XX animals, resulting in a variety of intersexual phenotypes. the tra-1(gf) alleles are dominant and have an opposite, feminizing effect. Six out of 22 can transform XO animals into fertile females or hermaphrodites, whereas the remainder cause partial feminization. All 22 transform XX animals into fertile females. Limited intragenic mapping indicates that the gene is large and that gf alleles map to a location different from lf alleles. The results suggest that the tra-1 gene has several roles in wild-type sexual development. First, tra-1 activity dictates female, as opposed to male, development in all nongonadal tissues of XX animals. Second, tra-1 activity dictates female development in the somatic gonad of XX animals. Third, a high level of tra-1 activity may act to inhibit spermatogenesis in the XX germ line, thereby assisting the switch from spermatogenesis to oogenesis in the hermaphrodite. These three functions are all feminizing and specific to the XX animal; the gene also has minor functions in the XO animal, which are to assist normal male somatic gonad development and to promote abundant spermatogenesis in males. A low level of both spermatogenesis and oogenesis can occur in the absence of tra-1 activity. Both the function and the regulation of this gene must be complex.
Fis binding to the recombinational enhancer of the Hin DNA inversion system.
Authors: Bruist M F , Glasgow A C , Johnson R C , Simon M I
Published: June 2007

The recombinational enhancer of the Hin inversion system in Salmonella stimulates recombination in vitro 150-fold in the presence of the Escherichia coli host factor Fis. To gain an understanding of the roles of the enhancer and Fis in stimulating the Hin-mediated inversion reaction, we have used nuclease and chemical protection/interference studies and gel retardation assays to examine the interactions between Fis and the recombinational enhancer. These studies combined with mutational analysis defined the enhancer sequences required for Fis binding and function. Fis binds with different affinities to two domains within the enhancer sequence. The binding of Fis at each domain is independent of the occupancy of the other domain and appears to be to opposite faces of the DNA helix. These results support a model for the role of the recombinational enhancer in Hin-mediated inversion in which the interaction between Hin bound at recombination sites and Fis bound to each domain of the recombinational enhancer results in a structure with the proper alignment and topology to promote DNA inversion.
Telomeric DNA-protein interactions of Oxytricha macronuclear DNA.
Authors: Price C M , Cech T R
Published: June 2007

Telomeres of Oxytricha macronuclear chromatin exist as discrete nonnucleosomal DNA-protein complexes, each of which encompasses the terminal 100-150 bp of a macronuclear DNA molecule. We have used chemical and nuclease footprinting to examine the internal structure of these telomeric complexes. Remarkably salt-stable DNA-protein interactions result in methylation-protection of specific guanine residues in the 3'-terminal T4G4T4G4 tail. The methylation pattern seen in vivo and in isolated macronuclei is reconstituted in vitro when purified 55-kD and 43-kD telomere proteins are added to purified macronuclear DNA. Very different interactions are observed between protein and DNA within the region approximately 45-135 bp from the 5' terminus. The DNase I cleavage pattern indicates that this DNA lies on the outside surface of protein but is not part of a nucleosome. Our data suggest that the telomeric complexes have two structural domains characterized by their dissimilar DNA-protein interactions. We propose that functionally equivalent telomeres from other organisms could be accommodated in a similar telomeric chromatin structure.
Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions.
Authors: Kroos L , Kaiser D
Published: June 2007

Certain developmental mutants of Myxococcus xanthus can be complemented extracellularly by wild-type cells. These mutants behave as if they are defective in cell-cell interactions that are required for development. There may be several different interactions because the mutants belong to four extracellular complementation groups (A, B, C, and D). We report here that B- and C- mutations change the pattern of gene expression during Myxococcus development as detected by transcriptional fusions to lacZ mediated by Tn5 lac. The mutant C locus reduced or abolished developmental beta-galactosidase expression from 15 lac fusions that normally begin to be expressed in wild-type cells after 6 hr of development. Expression of these C-dependent lac fusions was restored to C- mutants by adding wild-type cells. The C- mutation did not affect the expression of 10 lac fusions that normally begin to be expressed before 6 hr of development, indicating that the C-mediated cell-cell interaction is required beginning at about 6 hr of development. Cells require the B+ function very early in development because a B- mutation reduced or abolished developmental beta-galactosidase expression from all 26 lac fusions tested, including some that normally begin to be expressed at the onset of development. In a C- mutant and in a B- mutant, some lac fusions responded with reduced beta-galactosidase expression, whereas other fusions, which would normally begin beta-galactosidase expression at about the same time during development, expressed no beta-galactosidase, indicating that developmental genes within a given temporal class display different sensitivities to the absence of cell-cell interactions. Requirements for B+ and C+ function, as well as the previously described A+ function, appear to lie on the same developmental pathway.
Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.
Authors: Gounari F , Banks G R , Khazaie K , Jeggo P A , Holliday R
Published: June 2007

We report the isolation and characterization of a mammalian strain (tsm) that has a temperature-sensitive mutation in DNA methylation. The isolation procedure was based on the observation that treatment of a CHO TK- MT- cell line with demethylating agents introduces up to 46% demethylation, resulting in phenotypic reversion and transcriptional activation of the thymidine kinase (TK) and metallothionein (MT) genes at frequencies ranging from 1% to 59%. Seven thousand individual colonies from an EMS-mutagenized CHO TK- MT- population were screened for spontaneous reversion to TK+ phenotype after treatment at 39 degrees C. Successful isolates were subsequently examined for MT+ reversion. A single clone (tsm) was obtained that showed temperature-dependent reactivation of both TK and MT genes at frequencies of 7.2 X 10(-4) and 6 X 10(-4), respectively. The tsm cells were viable at 39 degrees C and showed no increased mutation frequency. Reactivation correlated with transcriptional activation of the respective genes, whereas backreversion to the TK- phenotype was associated with transcriptional inactivation. TK- backrevertants were reactivable again with demethylating agents. Although demethylation in tsm cells was not detectable by HPLC, Southern blot analysis revealed that reactivants, irrespective of their mode of generation, showed specific demethylation of both TK and MT genes. Also, after about 150 cell generations after treatment, reactivants from both temperature-induced tsm and cells exposed to demethylating agents gained 60% and 23%, respectively, in 5-methylcytosine (5mC). It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation. The further characterization of this and other mammalian mutants should help to clarify the physiological role of DNA methylation, as well as its regulation.
The major late transcription factor binds to and activates the mouse metallothionein I promoter.
Authors: Carthew R W , Chodosh L A , Sharp P A
Published: June 2007

Human (HeLa) cells contain a protein, MLTF, which specifically binds to a DNA sequence in the adenovirus 2 major late promoter and activates transcription of that promoter. The presence of MLTF in uninfected cells suggests that this factor contributes to the transcription of some cellular genes. We find that MLTF binds in a sequence-specific manner to the 5'-flanking region of the mouse metallothionein I (mMTI) gene. Binding was localized between -101 and -94 (relative to the initiation site at +1) by DNA-binding gel electrophoresis assay and DNA methylation interference analysis. As in adenovirus, binding occurred in a region containing the sequence CPuCGTGAC. Deletion of this sequence both eliminated the binding of MLTF and produced a fourfold reduction in transcriptional efficiency in vitro. In contrast to the intact promoter, transcription from the deletion mutant promoter was not stimulated by addition of purified MLTF to an in vitro reconstituted reaction. These results suggest that MLTF contributes to the transcription of cellular genes.
Tubulogenesis in Drosophila: a requirement for the trachealess gene product.
Authors: Isaac D D , Andrew D J
Published: June 2007

The trachealess (trh) gene of Drosophila is required for embryonic tube formation. In trh mutants, tube-forming cells of the salivary gland, trachea, and filzkörper fail to invaginate to form tubes and remain on the embryo surface. We identified a P-element insertion that disrupts trh function and used the insert to clone and characterize trh. trh is expressed in the salivary duct, trachea, and filzköper primordia, and expression persists in these cells throughout embryogenesis. trh expression in the salivary duct is controlled by the homeotic gene, Sex combs reduced (Scr), and by another salivary gland gene, fork head (fkh). trh is homologous to two transcription factors: the human hypoxia-inducible factor-1 alpha and the Drosophila Single-minded protein.
DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.
Authors: Gaal T , Ross W , Blatter E E , Tang H , Jia X , Krishnan V V , Assa-Munt N , Ebright R H , Gourse R L
Published: June 2007

The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.
Hlx homeo box gene is essential for an inductive tissue interaction that drives expansion of embryonic liver and gut.
Authors: Hentsch B , Lyons I , Li R , Hartley L , Lints T J , Adams J M , Harvey R P
Published: June 2007

The divergent murine homeo box gene Hlx is expressed in restricted hematopoietic cell types and, during embryogenesis, prominently in visceral mesenchyme of the developing liver, gall bladder, and gut. Targeted disruption of the gene has now established that it plays a key role in visceral organogenesis. Embryos homozygous for the mutation died around embryonic day 15 with anemia and severe hypoplasia of the liver and gut. Liver ontogeny commenced normally with formation of the liver diverticulum and differentiation of hepatocytes, but the organ failed to expand and reached only 3% of normal size. The apparent liver hypoplasia was not associated with a notable increase in apoptotic cells. Gut development also began normally, but the intestines failed to undergo extensive elongation and looping and reached only a quarter of normal length. The anemia resulted from a deficiency in the fetal form of hematopoiesis, which occurs in the liver, but no intrinsic defect in Hlx-/- hematopoietic cells was observed in vitro, and liver-derived Hlx-/- hematopoietic stem cells that were transplanted to irradiated normal mice could fully reconstitute hematopoiesis. The impaired fetal hematopoiesis therefore reflects insufficient support function provided by the minute liver. Hlx is normally expressed in visceral mesenchyme lying adjacent to the developing liver and gut epithelia affected by the mutation, but not in the epithelia themselves. Hence, Hlx regulates a mesenchymal-epithelial interaction that drives a vital growth phase in visceral organogenesis. Moreover, because mutation of Hlx blocked liver growth but not its specification, early morphogenesis, or differentiation, development of this organ appears to occur by step-wise inductive interactions under separate genetic control.
trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila.
Authors: Wilk R , Weizman I , Shilo B Z
Published: June 2007

The embryonic tracheal system in Drosophila develops from placodes of precursor cells on the ectoderm. A transcription factor of the basic helix-loop-helix (bHLH)-PAS family, which is expressed in the nuclei of the tracheal cells throughout development, was identified. The protein shows the highest degree of homology to the Single-minded (Sim) protein. The transcript represents the previously identified trachealess (trh) locus, essential for tracheal development. Ectopic expression of trh leads to generation of extra tracheal pits and branches and to the expression of tracheal markers by patches of ectodermal cells. The expression of trh is consistent with a biphasic mode of transcriptional regulation. Expression is first induced by exogenous cues and is subsequently autoregulated. trh is also expressed and required in the posterior spiracles and the salivary gland ducts. The role of Trachealess in the formation of several tubular tissues in the embryo suggests that it may induce a general fate of branched tubular structures of epithelial origin.
RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila.
Authors: Du W , Vidal M , Xie J E , Dyson N
Published: June 2007

Genetic studies have shown that cyclin E and dE2F are critical regulators of S-phase entry during Drosophila embryogenesis. Whereas the ectopic expression of cyclin E activates dE2F-dependent transcription, it has been proposed that cyclin E does not act directly on dE2F but targets a negative regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins (pRB, p107, and p130) that associate with E2F in humans; however, extensive efforts have failed to find such homologs in Drosophila. We have developed a two-hybrid approach that allows transcription activators to be used as bait for interacting proteins. From a screen using Drosophila E2F (dE2F and dDP) as bait, we identified a novel gene, RBF. RBF combines several of the structural features of pRB, p107, and p130, suggesting that it may have evolved from a common ancestor to the three human genes. RBF associates with dE2F and dDP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF was phosphorylated by a cyclin E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF plays a critical role in the regulation of cell proliferation in Drosophila and show that analogous pathways regulate S-phase entry in a diverse range of species.
Promoter specificity mediates the independent regulation of neighboring genes.
Authors: Merli C , Bergstrom D E , Cygan J A , Blackman R K
Published: June 2007

Although enhancers can exert their influence over great distances, their effect is generally limited to a single gene. To discern the mechanism by which this constraint can he mediated, we have studied three neighboring Drosophila genes: decapentaplegic (dpp), SLY1 homologous (Slh) and out at first (oaf). Several dpp enhancers are positioned close to Slh and oaf, and yet these genes are unaffected by the dpp elements. However, when a transposon is located within the oaf gene, the dpp enhancers activate the more distant transposon promoters while still ignoring the closer Slh and oaf start sites. To test whether this promoter specificity accounts for the regulatory autonomy normally found for the three genes, we used in vivo gene targeting to replace the oaf promoter with a dpp-compatible one in an otherwise normal chromosome. Strikingly, this chimeric gene is now activated by the dpp enhancers. Thus, the properties of the promoters themselves are sufficient to mediate the autonomous regulation of genes in this region.
Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene.
Authors: Lamonerie T , Tremblay J J , Lanctôt C , Therrien M , Gauthier Y , Drouin J
Published: June 2007

The pituitary gland contains six distinct hormone-producing cell types that arise sequentially during organogenesis. The first cells to differentiate are those that express the pro-opiomelanocortin (POMC) gene in the anterior pituitary lobe. The other lineages, which appear later, include cells that are dependent on the POU factor Pit-1 and another POMC-expressing lineage in the intermediate pituitary lobe. Using AtT-20 cells as a model for early expression of POMC in the anterior pituitary, we have defined a regulatory element conferring cell specificity of transcription and cloned a cognate transcription factor. This factor, Ptx1 (pituitary homeo box 1), contains a homeo box related to those of the anterior-specific genes bicoid and orthodenticle in Drosophila, and Otx-1 and Otx-2 in mammals. Ptx1 activates transcription upon binding a sequence related to the Drosophila bicoid target sites. Ptx1 is the only nuclear factor of this DNA-binding specificity that is detected in AtT-20 cells, and it is expressed at high levels in a subset of adult anterior pituitary cells that express POMC. However, Ptx1 is expressed in most cells of Rathke's pouch at an early time during pituitary development and before final differentiation of hormone-producing cells. Thus, Ptx1 may have a role in differentiation of pituitary cells, and its early expression pattern suggests that it may have a role in pituitary formation. In the adult pituitary gland, Ptx1 appears to be recruited for cell-specific transcription of the POMC gene.
A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae.
Authors: Ma X J , Lu Q , Grunstein M
Published: June 2007

In a genetic screen for second-site mutations that are lethal in combination with a deletion of the amino terminus of histone H3, we have uncovered three new gene products that regulate the Saccharomyces cerevisiae Swe1 kinase. The Swe1 protein kinase phosphorylates tyrosine residue 19 of Cdc28 and inhibits its activity. One histone synthetic-lethal gene, HSL1, encodes a putative protein kinase that has high sequence and functional homology to fission yeast cdr1/nim1, an inhibitory kinase of wee1. Another gene, HSL7, is a novel negative regulator of Swe1 function. Sequences similar to Hsl7 exist in Caenorhabditis elegans and humans. In addition, we have isolated a dosage-dependent suppressor, OSS1, of hsl1 and hsl7. OSS1 is important for the transcriptional repression of SWE1 and CLN2 in G2. Mutations in HSL1 and HSL7 therefore cause hyperactivity of the Swe1 kinase, which in turn decreases mitotic Cdc28 kinase activity. Moreover, HSL5 is identical to CDC28, further suggesting that it is the decreased Cdc28 kinase activity in these hsl mutants that causes lethality in the histone mutant background. Because neither HSL1 nor HSL7 is essential in yeast, and histone transcription is unaffected by the hsl5/cdc28 mutation, it is unlikely that synthetic lethality results from reduced transcription of HSL1 and HSL7 caused by histone mutations, or from reduced histone transcription when Cdc28 kinase activity is compromised. We suggest that these cell cycle regulators function in a pathway upstream of both histones H3 and H4, thereby modulating histone function in the cell cycle.
Coordinate activation of c-Src by SH3- and SH2-binding sites on a novel p130Cas-related protein, Sin.
Authors: Alexandropoulos K , Baltimore D
Published: June 2007

To understand how protein-protein interactions mediated by the Src-SH3 domain affect c-Src signaling, we screened for proteins that interact with the Src-SH3. We found a novel protein, Sin (Src interacting or signal integrating protein), that binds to Src-SH3 with high affinity, contains numerous tyrosine residues in configurations suggestive of SH2-binding sites, and is related to the v-Src substrate p130Cas. In cotransfection assays, a small fragment of Sin retaining the Src-SH3-binding site and one tyrosine-containing motif induced c-Src activation as measured by a transcriptional reporter. Phosphorylation of the peptide on tyrosine by c-Src, as a consequence of Src-SH3 binding, was necessary for its stable interaction with c-Src in vivo and for transcriptional activation. Phosphorylation of multiple tyrosine-containing motifs found on Sin correlated with c-Crk and cellular phosphoprotein binding to Sin as well as increased c-Src activity. These data suggest that (1) SH2 and SH3 ligand sites on Sin cooperatively activate the signaling potential of c-Src, (2) Sin acts as both an activator and a substrate for c-Src, and (3) phosphorylated Sin may serve as a signaling effector molecule for Src by binding to multiple cellular proteins.
Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development.
Authors: Kratochwil K , Dull M , Farinas I , Galceran J , Grosschedl R
Published: June 2007

Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the formation of organs that depend on epithelial-mesenchymal tissue interactions. In this study we have recombined epithelial and mesenchymal tissues from normal and LEF-1-deficient embryos at different stages of development to define the LEF-1-dependent steps in tooth and whisker organogenesis. At the initiation of organ development, formation of the epithelial primordium of the whisker but not tooth is dependent on mesenchymal Lef1 gene expression. Subsequent formation of a whisker and tooth mesenchymal papilla and completion of organogenesis require transient expression of Lef1 in the epithelium. These experiments indicate that the effect of Lef1 expression is transmitted from one tissue to the other. In addition, the finding that the expression of Lef1 can be activated by bone morphogenetic protein 4 (BMP-4) suggests a regulatory role of this transcription factor in BMP-mediated inductive tissue interactions.
Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs.
Authors: Asano M , Nevins J R , Wharton R P
Published: June 2007

Previous experiments suggest that a key event in the commitment of cultured mammalian cells to entering S phase is a rise in activity of the transcription factor E2F. In this report, we study the role of Drosophila E2F in imaginal disc cells in vivo, by examining the distribution of the endogenous protein and studying the consequences of ectopic E2F expression. First, we find that endogenous E217 falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated G1-S-transition in the eye disc. Second, we find that ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Third, we find that ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Although we do not know the fate of these cells, we suspect that ultimately they are killed by ectopic E2F. Taken together, our results show that an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase. Furthermore, they suggest that the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis.
Activator-dependent regulation of transcriptional pausing on nucleosomal templates.
Authors: Brown S A , Imbalzano A N , Kingston R E
Published: June 2007

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse genes, including human c-myc and c-fos, some HIV genes, and the Drosophila heat shock loci. To characterize the mechanisms that regulate pausing, we have established an in vitro system using the human hsp7O gene. We demonstrate that nucleosome formation increases by >100-fold the duration of a transcriptional pause on the human hsp7O gene in vitro at the same location as pausing is observed in vivo. Readthrough of this pause is increased by an activator that contains the human heat shock factor 1 (HSF1) transcriptional activation domains. Maximal effect of the activator requires that the system be supplemented with fractions that have hSWI/SNF activity, which has been shown previously to alter nucleosome structure. No significant readthrough is observed in the absence of activator, and neither the activator nor the hSWI/SNF fraction affected elongation on naked DNA; therefore, these results suggest that an activator can cause increased readthrough of promoter-proximal pausing by decreasing the inhibitory effect of nucleosomes on transcriptional elongation.
Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control.
Authors: Patra D , Dunphy W G
Published: June 2007

The small Suc1/Cks protein is a ubiquitous subunit of Cdk/cyclin complexes, but its precise function has remained unclear. We have isolated a Xenopus homolog, Xe-p9, of the Suc1/Cks protein by virtue of its ability to rescue a fission yeast mutant that enters mitosis prematurely. To assess its functional role in cell cycle control, we have both overexpressed p9 in Xenopus egg extracts and immunodepleted the protein from these extracts. We found that addition of recombinant His6-p9 to egg extracts results in a pronounced delay of mitosis that can be attributed to an inhibition of the tyrosine dephosphorylation of the inactive Cdc2/cyclin B complex. In immunodepletion studies, we observed that the consequences of removing p9 from egg extracts depend on the stage of the cell cycle. Specifically, in the case of interphase extracts, the removal of p9 abolishes the entry into mitosis as a result of a failure in the activation of the Cdc2/cyclin B complex by tyrosine dephosphorylation. Furthermore, mitotic extracts lacking p9 fail to exit mitosis because of a defect in the destruction of cyclin B. Collectively, these results indicate that p9 has multiple essential roles in the cell cycle by governing the interaction of the Cdc2/cyclin B complex with both positive and negative regulators.