bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae.
Authors: Wotton D , Shore D
Published: June 2007

The Saccharomyces cerevisiae Rap1 protein binds with high affinity to sites within the poly(C(1-3)A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing. Rap1p initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression. This same domain of Rap1p also negatively regulates telomere elongation, through an unknown mechanism. We have identified a new Rap1-interacting factor (Rif2p) that plays a role in telomere length regulation. Rif2p has considerable functional similarities with a Rap1p-interacting factor (Rif1p) identified previously. Mutations in RIF1 or RIF2 (unlike mutations in the silencing genes SIR3 and SIR4) result in moderate telomere elongation and improved telomeric silencing. However, deletion of both RIF1 and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Rap1p. In addition, overexpression of either RIF1 or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Rap1p carboxyl terminus. Finally, we show that Rif1p and Rif2p can interact with each other in vivo. These results suggest that telomere length regulation is mediated by a protein complex consisting of Rif1p and Rif2p, each of which has distinct regulatory functions. One role of Rap1p in telomere length regulation is to recruit these proteins to the telomeres.
New functional activities for the p21 family of CDK inhibitors.
Authors: LaBaer J , Garrett M D , Stevenson L F , Slingerland J M , Sandhu C , Chou H S , Fattaey A , Harlow E
Published: June 2007

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a), respectively, mostly because of a decrease in K(off). At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.
Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1.
Authors: Perkins C P , Mar V , Shutter J R , del Castillo J , Danilenko D M , Medlock E S , Ponting I L , Graham M , Stark K L , Zuo Y , Cunningham J M , Bosselman R A
Published: June 2007

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.
A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.
Authors: Min H , Turck C W , Nikolic J M , Black D L
Published: June 2007

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.
GATA4 transcription factor is required for ventral morphogenesis and heart tube formation.
Authors: Kuo C T , Morrisey E E , Anandappa R , Sigrist K , Lu M M , Parmacek M S , Soudais C , Leiden J M
Published: June 2007

Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development. In the studies described in this report we have used gene targeting to produce GATA4-deficient mice. Homozygous GATA4-deficient (GATA4-/-) mice died between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern. This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes was not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/- -C57BL/6 chimeric mice. Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis.
mirror encodes a novel PBX-class homeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye.
Authors: McNeill H , Yang C H , Brodsky M , Ungos J , Simon M A
Published: June 2007

The Drosophila eye is composed of dorsal and ventral mirror-image fields of opposite chiral forms of ommatidia. The boundary between these fields is known as the equator. We describe a novel gene, mirror (mrr), which is expressed in the dorsal half of the eye and plays a key role in forming the equator. Ectopic equators can be generated by juxtaposing mrr expressing and nonexpressing cells, and the path of the normal equator can be altered by changing the domain of mrr expression. These observations suggest that mrr is a key component in defining the dorsal-ventral boundary of tissue polarity in the eye. In addition, loss of mrr function leads to embryonic lethality and segmental defects, and its expression pattern suggests that it may also act to define segmental borders. Mirror is a member of the class of homeoproteins defined by the human proto-oncogene PBX1. mrr is similar to the Iroquois genes ara and caup and is located adjacent to them in this recently described homeotic cluster.
Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation.
Authors: Kaya G , Rodriguez I , Jorcano J L , Vassalli P , Stamenkovic I
Published: June 2007

CD44 is a broadly distributed polymorphic glycoprotein that serves as the principal cell-surface receptor for hyaluronate. Although CD44-mediated cell interaction with hyaluronate has been implicated in a variety of physiologic events, including cell-cell and cell-substrate adhesion, cell migration, proliferation, and activation, as well as hyaluronate uptake and degradation, the biologic role of CD44 in vivo in various tissues remains to be determined. In the present work we have developed transgenic mice that express an antisense CD44 cDNA driven by the keratin-5 promoter. These mice lack detectable CD44 expression in skin keratinocytes and corneal epithelium and display abnormal hyaluronate accumulation in the superficial dermis and corneal stroma, distinct morphologic alterations of basal keratinocytes and cornea, and defective keratinocyte proliferation in response to mitogen and growth factors. These alterations are reflected by a decrease in skin elasticity, impaired local inflammatory response and tissue repair, delayed hair regrowth, and failure of the epidermis to undergo hyperplasia in response to carcinogen. Our observations indicate that two major functions of CD44 in skin are the regulation of keratinocyte proliferation in response to extracellular stimuli and the maintenance of local hyaluronate homeostasis.
Requirement for Drosophila 14-3-3 zeta in Raf-dependent photoreceptor development.
Authors: Kockel L , Vorbrüggen G , Jäckle H , Mlodzik M , Bohmann D
Published: June 2007

Based on biochemical and functional data obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated in a number of different signal transduction processes, in particular in the signal-dependent activation of protein kinases. We performed a functional analysis of 14-3-3 in a multicellular organism, initiated by the cloning of a 14-3-3 zeta homolog of Drosophila melanogaster, termed D14-3-3 zeta. D14-3-3 zeta transcripts are strongly enriched in the developing central nervous system. In addition, they are predominantly expressed in the region posterior to the morphogenetic furrow of the eye imaginal disc where cells differentiate as photoreceptors. In these cells D14-3-3 zeta is localized apically. Both the expression pattern and the subcellular localization are consistent with the proposed function of 14-3-3 proteins in Ras/Raf/MAPK signaling. D14-3-3 zeta mutant analysis combined with rescue experiments involving gain-of-function alleles of Raf and Ras indicate that D14-3-3 zeta is an essential component of the Raf/Ras signaling pathway and necessary for photoreceptor differentiation. It acts upstream of Raf and downstream of Ras.
Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning.
Authors: Glaser P , Sharpe M E , Raether B , Perego M , Ohlsen K , Errington J
Published: June 2007

The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.
A specific 15-bp TATA box promoter element is required for expression of a herpes simplex virus type 1 late gene.
Authors: Homa F L , Glorioso J C , Levine M
Published: June 2007

The herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene is a true late or gamma 2 gene in that its expression shows a strict requirement for viral DNA replication. Elements required for regulated expression of this gene were previously shown to consist of the gC TATA box, transcription start site and a large portion of the leader sequence of the gC gene. In this paper we show that transcription of the gC gene requires a 15-bp sequence, GGGTATAAATTCCGG, which contains the gC TATA box. This sequence contains specific promoter elements because replacement of this sequence with either the TATA box of the HSV-1 early thymidine kinase (tk) gene or two random TATA-like elements results in a transcriptionally inactive gC gene. In addition, we show that temporal expression of HSV beta and gamma genes at early and late times during infection are controlled by separate and distinct regulatory elements; regulatory signals distal to the TATA box are needed for early expression, whereas a gC-like TATA box is needed for late expression. These signals were identified by construction of a chimeric HSV gene that contained the distal control signals of the beta tk gene fused upstream of the TATA sequence of the gamma 2 gC gene. When RNA was isolated at various times postinfection from cells infected with a virus whose genome contained this chimeric tk-gC gene, synthesis of gC mRNA showed both early and late kinetics.
5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements.
Authors: Siliciano P G , Guthrie C
Published: June 2007

Using a strategy of compensatory nucleotide changes between yeast U1 and a 5' splice site, we have analyzed the contribution of base-pairing to the efficiency and fidelity of pre-mRNA splicing in vivo. Watson-Crick base-pairing interactions with U1 can be demonstrated at intron positions 1 and 5 but not at position 4. Moreover, restoration of the ability to pair with U1 is not sufficient to restore activity in the second step of splicing to intron position 1 mutants. Finally, in contrast to recent observations in mammalian systems, we find that the precise position of 5' splice site cleavage is not determined solely by the base-pairing interaction with U1. Rather, the presence of a G residue at position 5 is required for the correct localization of the nucleolytic event. Taken together, these results indicate that the demands for 5' splice site selection and utilization are more complex than a simple maximization of Watson-Crick interactions with U1.
The role of the mammalian branchpoint sequence in pre-mRNA splicing.
Authors: Reed R , Maniatis T
Published: June 2007

We show that base substitutions in the mammalian branchpoint sequence (BPS) YNCUGAC dramatically reduce the efficiency of pre-mRNA splicing in vitro and alter 3' splice-site selection in vivo. Contrary to current dogma that an adenine residue at the appropriate distance from the 3' splice site is the primary determinant of lariat formation, we find that many mutations in the BPS virtually abolish splicing even though the position of this adenine is unchanged. Comparison of the analogous single-base changes in the mammalian and yeast BPSs revealed similar relative effects on splicing efficiency. However, in contrast to yeast, mammalian branchpoint mutations that decrease splicing efficiency severely do not prevent spliceosome assembly. Thus, mutations in the mammalian BPS appear to uncouple spliceosome assembly from cleavage at the 5' splice site and lariat formation.
Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes.
Authors: Bektesh S , Van Doren K , Hirsh D
Published: June 2007

Several different mRNAs from Caenorhabditis elegans contain the same 22-nucleotide leader sequence at their 5' ends that is acquired in a trans-splicing reaction. About 10 to 15% of the major proteins are translated from mRNAs that contain the spliced leader, among them two ribosomal proteins, ubiquitin, GAPDH, a heat shock protein (hsp70a), and three actins. The same spliced leader sequence is present in mRNAs isolated from nematodes from several different genera; but it is not present in mRNAs from other organisms. The spliced leader is encoded as a spliced leader (SL) RNA about 100 nucleotides long. The gene for the SL RNA is located in the 5S rDNA repeat in C. elegans; however, this association with the 5S repeat is not preserved in other genera. The 22-nucleotide spliced leader sequence is conserved in three genera of nematodes.
Expression of a histone H1-like protein is restricted to early Xenopus development.
Authors: Smith R C , Dworkin-Rastl E , Dworkin M B
Published: June 2007

Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone H1, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone H1 mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone H1 3' noncoding consensus sequence involved in RNA processing.
Myosin heavy-chain mutations that disrupt Caenorhabditis elegans thick filament assembly.
Authors: Bejsovec A , Anderson P
Published: June 2007

We have investigated Caenorhabditis elegans mutants in which altered unc-54 myosin heavy-chain protein interferes with assembly of thick myofilaments. These mutants have a dominant, muscle-defective phenotype, because altered myosin heavy-chain B (MHC B), the product of the unc-54 gene, disrupts assembly of wild-type MHC B. The mutant MHC B also interferes with assembly of wild-type myosin heavy-chain A (MHC A), the product of another MHC gene expressed in body-wall muscle cells. Because of disrupted MHC A assembly, dominant unc-54 mutants also exhibit a recessive-lethal phenotype. Dominant unc-54 mutations are missense alleles, and the defects in thick filament assembly result from mutant protein that is of normal molecular weight. Accumulation of mutant MHC B in amounts as little as 2% of wild-type levels is sufficient to disrupt assembly of both wild-type MHC A and MHC B. Dominant unc-54 mutations occur at remarkably high frequency following ethylmethane sulfonate (EMS) mutagenesis; their frequency is approximately equal to that of recessive, loss-of-function mutations. This unusually high gain-of-function frequency implies that many different amino acid substitutions in the myosin heavy-chain B protein can disrupt thick filament assembly.
Mechanisms underlying generation of gradients in gene expression within the intestine: an analysis using transgenic mice containing fatty acid binding protein-human growth hormone fusion genes.
Authors: Sweetser D A , Birkenmeier E H , Hoppe P C , McKeel D W , Gordon J I
Published: June 2007

The intestine is lined by a continuously regenerating epithelium that maintains gradients in 'liver' fatty acid binding protein (L-FABP) gene expression along its horizontal and vertical axes, i.e., from duodenum to colon and from crypt to villus tip. To identify cis-acting DNA sequences responsible for these regional differences, we linked portions of the L-FABP gene's 5' nontranscribed region to the human growth hormone (hGH) gene and examined hGH expression in transgenic mice. Nucleotides -596 to +21 of the rat L-FABP gene correctly directed hGH expression to enterocytes and hepatocytes. However, anomalous expression was observed in small intestinal crypts, colon, and renal proximal tubular epithelial cells. Addition of nucleotides -4000 to -597 of the L-FABP gene, in either orientation, suppressed renal hGH expression and restored a nearly normal horizontal, but not a vertical, hGH gradient in the intestine. Thus, horizontal gradients of gene expression within the intestine can be maintained by orientation-independent, cis-acting suppressor elements.
Analysis of the lethal interaction between the prune and Killer of prune mutations of Drosophila.
Authors: Biggs J , Tripoulas N , Hersperger E , Dearolf C , Shearn A
Published: June 2007

The third-chromosome mutation Killer of prune (K-pn) causes no phenotype by itself, but causes lethality in individuals homozygous for the nonlethal X-chromosome mutation prune (pn). We have recovered 12 gamma-ray-induced revertants of Killer of prune. All of the revertants fail to complement a recessive cell lethal mutation in the abnormal wing discs (awd) gene. We present evidence that Killer of prune is a mutation in the awd gene. First, revertant awdKR14 leads to reduced accumulation of the awd gene product, but does not affect flanking genes. Second, when a copy of the awd gene is cloned from Killer of prune homozygous flies and injected into embryos, transformants express the lethal interaction with prune. In individuals of the genotype pn; awdK-pn/awd+ the awd mRNA is present at normal levels but the awd polypeptide fails to accumulate. The absence of the awd gene product in such individuals is the cause of death. Although the awd polypeptide is a subunit of a cytoplasmic protein, its sequence is similar to subunit V of yeast cytochrome oxidase.
The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos.
Authors: Thomson J A , Solter D
Published: June 2007

Both a maternal and a paternal genomic contribution are necessary for completion of embryonic development in the mouse. Parthenogenetic embryos, with only a maternally inherited genome, and androgenetic embryos, with only a paternally inherited genome, fail to develop to term, and these two types of isoparental embryos fail in development in characteristic ways. In this paper we describe the construction of chimeras between single androgenetic, parthenogenetic, and gynogenetic blastomeres and normal eight-cell embryos. We allow the development of the chimeras to reach the late-gastrulating-stage embryo and then analyze the tissue distributions of the isoparental component. The isoparental embryos are derived from a transgenic mouse line carrying plasmid and mouse beta-globin sequences. The isoparental cells are detected in histological sections of chimeras by DNA-DNA in situ hybridization to the transgene, using a biotinylated DNA probe with an enzymatic detection system. We found strong tissue preferences for the androgenetic, parthenogenetic, and gynogenetic cells in chimeras. Androgenetic cells contributed strongly to all trophectoderm-derived tissue, with only a rare contribution to any tissues of the embryo proper, extraembryonic mesoderm, or extraembryonic endoderm. Parthenogenetic cells shared a developmental fate similar to gynogenetic cells, contributing to all tissues of the embryo proper and to the extraembryonic mesoderm, but only rarely to the extraembryonic endoderm or to any trophectoderm-derived tissues.
Changes in histone gene dosage alter transcription in yeast.
Authors: Clark-Adams C D , Norris D , Osley M A , Fassler J S , Winston F
Published: June 2007

Chromatin structure is believed to be important for a number of cellular processes, including transcription. However, the role of nucleosomes in transcription is not well understood. We have identified the yeast histone locus HTB1-HTB1, encoding histones H2A and H2B, as a suppressor of solo delta insertion mutations that inhibit adjacent gene expression. The HTA1-HTB1 locus causes suppression either when present on a high-copy-number plasmid or when mutant. These changes in HTA1-HTB1 after transcription of the genes adjacent to the delta insertions. On the basis of this result, we have examined the effects of increased and decreased histone gene dosage for all four yeast histone loci. From the types of histone gene dosage changes that cause suppression of insertion mutations, we conclude that altered stoichiometry of histone dimer sets can alter transcription in yeast.
Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes.
Authors: Rotenberg M O , Moritz M , Woolford J L
Published: June 2007

We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes. Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type. RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts. No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant. Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein. Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable. rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits. 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits. We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis.
Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin H1 gene during early embryogenesis.
Authors: Lai Z C , Maxson R , Childs G
Published: June 2007

Late histone H1-beta mRNA accumulates with the correct ontogenic pattern following microinjection of the cloned gene into fertilized sea urchin eggs. Sequences upstream of the gene encoding the sea urchin H1-beta protein contain both basal and developmentally regulated elements. One late H1-specific activator sequence (USE IV) is required for the accumulation of mRNA following the blastula stage of development. All late H1 genes also contain a highly conserved GC-rich sequence resembling a low-affinity binding site for the mammalian transcription factor Sp1 that is required for basal expression of the H1-beta gene at all stages of embryogenesis. When this GC-rich sequence (GGGCTG) is converted to a perfect core Sp1 sequence (GGGCGG), the H1-beta transcripts accumulate to much greater levels and their peak accumulation is shifted to the early blastula stage rather than late blastula and gastrula stages of development. Coincidently, early H1 genes, whose peak expression is also at the early blastula stage, all contain the same core consensus sequence (GGGCGG). Thus, both gene-specific activator sequences, as well as sequences that resemble sites for general transcription factors, may play a major role in determining the temporal patterns of gene expression during early embryogenesis.
Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.
Authors: Ho Y S , Mahoney M E , Wulff D L , Rosenberg M
Published: June 2007

The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.
TFIIIA binds with equal affinity to somatic and major oocyte 5S RNA genes.
Authors: McConkey G A , Bogenhagen D F
Published: June 2007

Current models for the differential control of expression of Xenopus somatic and oocyte 5S RNA genes suggest that an impaired ability to bind TFIIIA contributes to the inactivation of oocyte 5S RNA genes in somatic cells. The somatic 5S RNA gene is transcribed more efficiently than the major oocyte 5S RNA gene in S-150 extracts of mature oocytes. However, this differential transcription efficiency is not determined simply by the relative affinity for binding of a positive transcription factor, TFIIIA. We have compared the abilities of somatic, major oocyte, and minor oocyte 5S RNA genes to bind TFIIIA using both a standard footprint competition assay and an indirect DNase protection assay. This indirect DNase protection assay permits the direct comparison of TFIIIA binding to two templates in one reaction. Both assay methods indicate that the major oocyte 5S RNA gene and the somatic 5S RNA gene bind TFIIIA with equal affinity. As a further control, we have confirmed earlier work indicating that the minor oocyte gene binds TFIIIA with a reduced affinity. Binding of TFIIIA to these three 5S RNA genes results in a different pattern of protection of each gene. We suggest that slight differences in the contacts between TFIIIA and the 5' border of the control region influence the ability of additional transcription factors to bind to the TFIIIA:5S DNA complex.
Temperature-sensitive v-sea transformed erythroblasts: a model system to study gene expression during erythroid differentiation.
Authors: Knight J , Zenke M , Disela C , Kowenz E , Vogt P , Engel J D , Hayman M J , Beug H
Published: June 2007

The isolation and characterization of a temperature-sensitive mutant (ts1 S13) of the avian erythroblastosis virus, S13, is described. The temperature-sensitive lesion in ts1 S13 was identified as affecting the tyrosine kinase activity but not the plasma membrane localization of the ts1 S13 v-sea gene product. Erythroblasts transformed by ts1 S13 can be induced to synchronously differentiate into erythrocytes in an erythropoietin (EPO)-dependent fashion. Analysis of erythrocyte-specific gene expression in ts1 S13 erythroblasts reveals that the transformed, self-renewing erythroblasts obtained at permissive temperature already express all erythrocyte genes tested for, although at a low level. Upon differentiation induction, expression of erythrocyte-specific genes is not coordinately regulated but rather involves complex regulatory mechanisms that appear to be specific for the individual genes.
Cloning and analysis of the dec-1 female-sterile locus, a gene required for proper assembly of the Drosophila eggshell.
Authors: Hawley R J , Waring G L
Published: June 2007

Female-sterile mutations at the dec-1 (defective chorion-1) locus of Drosophila severely disrupt the organization of the eggshell late in oogenesis. Previous characterization of dec-1 mutations has correlated the defects with failure to accumulate an early eggshell protein that undergoes proteolytic cleavage during choriogenesis. To enable further study of the regulation and processing of dec-1 products, we have molecularly cloned the locus and characterized its transcripts. Chromosome jumping was used to isolate a deficiency breakpoint within the locus. Overlapping genomic clones from a wild-type library were then obtained, and a region including the dec-1 locus was identified by hybridization to cDNA probes complementary to RNA from stage 9-10 egg chambers. Analysis of genomic rearrangements associated with the locus verified its identity. Two transcripts from the locus have been identified and characterized using cDNA clones, RNase protection, and primer extension analyses. A 4.0-kb transcript accumulates maximally in stages 9-10, when the primary follicle cell protein associated with dec-1 mutations is synthesized. A second transcript of 5.8 kb, generated by alternative splicing, accumulates during stages 11-12. These results are discussed in light of previous analysis of dec-1 mutations.
Temporal and spatial relationships between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi tarazu, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins.
Authors: Carroll S B , DiNardo S , O'Farrell P H , White R A , Scott M P
Published: June 2007

The specification of segment number and identity in the Drosophila embryo requires the activity of several classes of genes that may be grouped according to the array of pattern elements that they control. Double-label immunofluorescence was used to simultaneously localize the products of genes representative of the pair-rule segmentation class (fushi tarazu), the segment polarity class (engrailed), and the homeotic class (Sex combs reduced, Antennapedia, and Ultrabithorax) of pattern-regulating genes. The temporal order of appearance of each class of proteins and the precise spatial relationships between the products of the different genes are described with single-cell resolution. Boundaries of gene expression, particularly the parasegmental boundaries, are established by early-acting genes such as fushi tarazu and subsequently respected by the expression patterns of later appearing gene products such as engrailed and Ultrabithorax, suggesting regulatory relationships between certain pairs of genes. In addition, the dynamic transitions observed in spatial relationship among the Sex combs reduced, Antennapedia, and Ultrabithorax homeotic protein patterns during the early period of embryogenesis may reflect cross-regulatory interactions among these genes. Finally, some cells contain a single homeotic product, whereas other cells simultaneously contain several, suggesting that certain cells may be determined by the combinatorial action of homeotic genes.
Constraints on spacing between transcription factor binding sites in a simple adenovirus promoter.
Authors: Wu L , Berk A
Published: June 2007

The adenovirus 2 E1B transcription unit has an extremely simple promoter consisting of a TATA box and a closely situated GC box. The interaction between the TATA box and the GC box was analyzed using insertion mutations which expand the distance between the two elements. We observed that the E1B promoter has an unusually rigid architecture. When the GC box, which is a binding site for transcription factor Sp1, was separated further from the TATA box than in the wild-type promoter, in vivo transcription quickly diminished to a level comparable to the elimination of the Sp1 site. Yet all the insertion mutants bound Sp1 factor in vitro with an affinity approximately equal to that of the wild-type promoter. From these results, we argue that Sp1 binding alone is not sufficient to stimulate transcription. The increased distance might disrupt direct contacts between Sp1 and transcription factors bound at the TATA box, contacts required for transcription stimulation by Sp1. The insertion mutations do not interfere with the transcription activation process mediated by the adenovirus large E1A protein or the pseudorabies virus immediate early protein.
SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits.
Authors: Toda T , Cameron S , Sass P , Wigler M
Published: June 2007

A new gene, SCH9, was isolated from Saccharomyces cerevisiae by its ability to complement a cdc25ts mutation. Sequence analysis indicates that it encodes a 90,000-dalton protein with a carboxy-terminal domain homologous to yeast and mammalian cAMP-dependent protein kinase catalytic subunits. In addition to suppressing loss of CDC25 function, multicopy plasmids containing SCH9 suppress the growth defects of strains lacking the RAS genes, the CYR1 gene, which encodes adenylyl cyclase, and the TPK genes, which encode the cAMP-dependent protein kinase catalytic subunits. Cells lacking SCH9 grow slowly and have a prolonged G1 phase of the cell cycle. This defect is suppressed by activation of the cAMP effector pathway. We propose that SCH9 encodes a protein kinase that is part of a growth control pathway which is at least partially redundant with the cAMP pathway.
The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.
Authors: Liu J K , Bergman Y , Zaret K S
Published: June 2007

In this paper we characterize the chromatin structure and nuclear proteins associated with different transcriptional states of the mouse serum albumin gene. We found the albumin gene to be transcribed in liver at rates 1000-fold or greater than in other tissues tested. We discovered seven DNase I hypersensitive sites encompassing the albumin gene only in liver chromatin, with strong hypersensitivity at the promoter and the enhancer, which is over 10 kb upstream. Using a gel retardation assay, we found a liver nuclear protein, or set of proteins, which binds specifically to DNA of a liver-specific hypersensitive site that maps 3.5 kb upstream, between the promoter and enhancer. Footprinting, heat insensitivity, and binding competition experiments indicate that the protein(s) have characteristics similar to a heat-stable, liver-abundant protein that binds to the albumin promoter and other enhancer and promoter sequences. Finally, we asked whether the liver-specific factors that cause DNase I hypersensitivity in vivo are present concurrently at the various sites in chromatin. We devised a simple new method to reveal that in liver, individual albumin genes are hypersensitive simultaneously at the promoter, the enhancer, and the -3.5-kb site. Thus, transcriptionally active albumin genes appear to contain tissue-abundant factors that are present at three widely spaced points in chromatin, yet at the same point in time. Similar factors binding simultaneously to at least two of these sites could create a specific structure in chromatin required for high-level albumin gene transcription.
Constitutive expression of c-fos antisense RNA blocks c-fos gene induction by interferon and by phorbol ester and reduces c-myc expression in F9 embryonal carcinoma cells.
Authors: Levi B Z , Ozato K
Published: June 2007

To address the role of c-fos proto-oncogene we constructed a plasmid that allows constitutive expression of RNA complementary to c-fos mRNA, and stably introduced this plasmid into F9 embryonal carcinoma cells. Some F9 clones expressing c-fos antisense RNA had a reduced basal level of c-fos mRNA, and were unable to induce a c-fos mRNA as well as its protein when stimulated with phorbol ester or with interferon (IFN). Nevertheless, the ability to induce major histocompatibility class I genes following IFN treatment was not impaired in these clones. Clones expressing c-fos antisense RNA grew as rapidly as control F9 cells, and underwent differentiation after retinoic acid treatment. Unexpectedly, constitutive expression of c-myc mRNA was reduced on average by 10-fold in clones expressing c-fos antisense RNA. However, expression of the p53 gene and heat shock gene hsp 70 was not affected in these clones, indicating the existence of a specific regulatory linkage between c-fos and c-myc genes. Cycloheximide treatment led to induction of a large amount of c-fos mRNA in clones expressing c-fos antisense RNA as well as in control F9 clones. The amount of c-fos antisense RNA was also increased by cycloheximide treatment. We postulate that c-fos antisense RNA blocks expression of the endogenous c-fos gene by accelerating the degradation of c-fos mRNA and that cycloheximide treatment interferes with this degradation.
Multiple factors are required for specific RNA cleavage at a poly(A) addition site.
Authors: Gilmartin G M , McDevitt M A , Nevins J R
Published: June 2007

An SP6 RNA containing the adenovirus 5 L3 poly(A) site is processed efficiently in a HeLa cell nuclear extract to generate correct 3' termini. Accurate 3' processing has also been demonstrated for the adenovirus E2A and SV40 early poly(A) sites, although these are processed less efficiently than the L3 site. Efficient cleavage at the poly(A) site requires the presence of a 5'-cap structure, as well as the RNA sequence motifs previously shown to be necessary for 3' processing in vivo, suggesting the presence and action of the appropriate factors in the nuclear extract. Fractionation of the nuclear extract has revealed a requirement for at least two distinct factors for cleavage at the L3 poly(A) site. One of these factors appears to possess an RNA component due to its sensitivity to micrococcal nuclease. The activity of this fraction is also sensitive to alpha-Sm monoclonal antibody, indicating the presence of an snRNP essential for the cleavage reaction. Additional factors are required for the subsequent polyadenylation reaction, indicating the involvement of a multicomponent complex in the processing of an RNA at the poly(A) site.
Nucleotide sequence and structure of the sevenless gene of Drosophila melanogaster.
Authors: Bowtell D D , Simon M A , Rubin G M
Published: June 2007

In the Drosophila melanogaster mutant sevenless, the R7 photoreceptor in each of the repeating units, or ommatidia, of the compound eye fails to form. We have determined the nucleotide sequence and structure of the sevenless transcription unit. Overlapping cDNA clones from an eye imaginal disc library were isolated, and these together with corresponding genomic regions were sequenced. The positions of the major and two minor transcription start sites were mapped. The gene encodes a putative cell-surface receptor, the unmodified form of which is predicted as 288 kD, bearing a cytoplasmic tyrosine kinase domain. Several structural features distinguish sevenless from other tyrosine kinase receptors, most notably the large size of its extracellular domain. Moreover, unlike the insulin or epidermal growth factor (EGF) receptors, a putative amino-terminal signal sequence does not immediately follow the initiating methionine codon. Rather, a 21-amino-acid hydrophobic sequence is found 56 amino acids after the likely initiating methionine codon, suggesting that this sequence may function as an amino-terminal anchor. If so, the sevenless protein would be expected to have its 2001-amino-acid extracellular domain anchored as a loop at either end in the membrane. Finally, we have generated a number of transformant lines that carry either a 16.3-kb genomic fragment that extends approximately 950 bp upstream of the transcription start sites or constructs in which a subset of the introns present in the genomic sevenless DNA have been removed. The degree of rescue of the sevenless mutant phenotype has been assayed in these lines using morphological and behavioral assays.
DNA methylation: a secondary event in globin gene switching?
Authors: Enver T , Zhang J W , Papayannopoulou T , Stamatoyannopoulos G
Published: June 2007

Changes in gamma-globin gene methylation accompany the fetal to adult globin switch in man. Using somatic cell hybrids made by fusing mouse erythroleukemia and human fetal erythroid cells, we asked whether methylation is a cause or a consequence of gamma-gene inactivation during development. These hybrids initially express human gamma-globin but switch with time in culture to adult globin gene production. In hybrids before the switch, the gamma-genes are unmethylated. After completion of the switch, the hybrids contain methylated gamma-globin genes. However, during the time that the gamma----beta switch is occurring, hybrids are found that no longer express gamma-globin, yet still possess unmethylated gamma-globin genes. This suggests that methylation is not a primary event in gamma-gene inactivation during human development.
Mutations that alter both localization and production of a yeast nuclear protein.
Authors: Silver P A , Chiang A , Sadler I
Published: June 2007

The first 74 amino acids of the yeast GAL4 gene product are sufficient to localize a GAL4-beta-galactosidase chimeric protein to the yeast nucleus. Chimeric proteins missing the first 74 GAL4 amino acids, but containing almost all of the rest of GAL4, are not localized to the nucleus and are expressed at higher levels than their nuclear counterparts. On this basis, point mutations within GAL4, which reduce nuclear localization and increase production of a normally nuclear GAL4-beta-galactosidase fusion protein, were isolated and sequenced. The effect of these mutations on the localization and expression of the intact GAL4 protein was examined. The degree to which the mutant proteins are excluded from the nucleus varies, but all mutations cause overproduction of the protein. Point mutations altering two of the six cysteine residues of the GAL4 putative 'zinc finger' abolish gene activation by intact GAL4; however, mutations in nearby residues have no effect on GAL4-dependent gene activation.