bluuebunny/crossref_metadata_2025 huggingface dataset:
Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors.
Authors: Lin R J , Lustig A J , Abelson J
Published: June 2007

We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2 delta spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2 delta spliceosome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.
Lineage and fate of each blastomere of the eight-cell sea urchin embryo.
Authors: Cameron R A , Hough-Evans B R , Britten R J , Davidson E H
Published: June 2007

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage.
Cell- and promoter-specific activation of transcription by DNA replication.
Authors: Grass D S , Read D , Lewis E D , Manley J L
Published: June 2007

To study the effects that DNA replication can exert on transcription in mammalian cells, we have analyzed transient expression from the adenovirus major late promoter contained on replicating and nonreplicating plasmids in several cell types. When a 100-bp fragment containing the late promoter was used to direct expression of the simian virus 40 (SV40) early region, efficient transcription could be detected that was only slightly enhanced when a functional origin of replication was included in the plasmid. In contrast with this, and with similar findings using related late promoter-containing plasmids, expression from this promoter was absolutely dependent on DNA replication when it was inserted in the region of SV40 DNA encoding the late mRNA 5' ends and expression was assayed in human HeLa cells and BSC-1 and COS-7 monkey cells. In contrast, transcription was totally independent of replication in human 293 cells. These results, which were not due to differences in template copy number, suggest that both cis- and trans-acting factors can influence a promoter's response to DNA replication and point to possible functional similarities between replication origins and transcriptional enhancers.
cis- and trans-acting sequences required for expression of simian virus 40 genes in mouse oocytes.
Authors: Chalifour L E , Wirak D O , Hansen U , Wassarman P M , DePamphilis M L
Published: June 2007

To determine the requirements for gene expression in mammalian germ cells, circular double-stranded simian virus 40 (SV40) DNA molecules containing deletions in sequences controlling transcription and replication were injected into the nucleus of mouse oocytes. Expression of large (T-Ag) and small (t-Ag) tumor antigens ("early gene products") required at least three GGGCGG boxes, but did not require either the origin of viral DNA replication (ori) or a TATA box. Expression of capsid antigen VP1 ("late gene products") required at least three GGGCGG boxes, sequences between nucleotides 197 and 273 in the 72-bp repeat region, and transactivation by T-Ag. These results are consistent with the requirements for expression of the same genes in differentiated mammalian cells. Surprisingly, however, the 72-bp repeats ("enhancer elements") that are required for expression of T-Ag and t-Ag genes in differentiated cells were not required in mouse oocytes. Similarly, expression of both the early and late genes was unaffected in mouse oocytes by the absence of either DNA replication or an intact ori sequence, components required for maximum expression of late genes in differentiated cells. Thus, mammalian oocytes effectively utilize promoters that are fully active in mammalian differentiated cells only when associated with either enhancer elements or DNA replication. Furthermore, requirements for expression of SV40 genes in mouse oocytes are distinctly different from those reported for Xenopus oocytes. This suggests that caution should be exercised when extrapolating conclusions drawn from experiments with amphibian germ cells to mammalian germ cells.
GAL1-GAL10 divergent promoter region of Saccharomyces cerevisiae contains negative control elements in addition to functionally separate and possibly overlapping upstream activating sequences.
Authors: West R W , Chen S M , Putz H , Butler G , Banerjee M
Published: June 2007

The upstream activating sequence (UASG) of the adjacent and divergently transcribed GAL1 and GAL10 promoters of Saccharomyces cerevisiae regulates the induction of the corresponding genes in response to the presence of galactose. We constructed chimeric yeast promoters in which a different UAS, UASC from the iso-1-cytochrome c (CYC1) gene of S. cerevisiae, was fused at different locations upstream of GAL1 (UASC-GAL1 promoters) or GAL10 (UASC-GAL10 promoters) and used to monitor the activity of UASG in cells grown in the presence or absence of galactose. Though the CYC1 promoter is fully induced in yeast grown in glycerol medium, UASC-GAL chimeric promoters containing UASG were repressed as much as 400-fold (UASC-GAL1) or 1350-fold (UASC-GAL10) in this growth medium. Several distinct portions of the GAL1-GAL10 divergent promoter region blocked the UASC-induced expression of the GAL1 and GAL10 promoters, whereas others did not, suggesting that several distinct negative control elements are present that may repress transcription of GAL1 and GAL10 in the absence of galactose. The approximate locations of these negative control elements were delimited to sites adjacent to or possibly overlapping the sites at which the positive control protein GAL4 binds in UASG. Deletion derivatives of GAL4 that fail to induce transcription from the wild-type GAL promoters but retain the DNA binding domain significantly derepressed the expression of the UASC-GAL chimeric promoters. These results, combined with those of earlier studies, suggest the possibility that GAL4 normally induces transcription of GAL1 and GAL10 by blocking the activity of these negative control elements, in addition to stimulating transcription by a mechanism of positive control.
Identification of factors that interact with the E1A-inducible adenovirus E3 promoter.
Authors: Hurst H C , Jones N C
Published: June 2007

We have investigated the E1A-inducible E3 promoter of adenovirus type 5 with respect to its ability to bind specific nuclear proteins. Four distinct nucleoprotein-binding sites were detected, located between positions-7 to -33, -44 to -68, -81 to -103, and -154 to -183, relative to the E3 cap site. These sites contain sequences previously shown to be functionally important for efficient E3 transcription. No major qualitative or quantitative differences were found in the binding pattern between nucleoprotein extracts prepared from uninfected or adenovirus-infected HeLa cells. Competition experiments suggest that the factors binding to the -154 to -183 and -81 to -103 sites are the previously identified nucleoproteins, NF1 and AP1, respectively. The factor binding to the -44 to -68 site, which we term ATF, also interacts with other E1A-inducible promoters and is very similar and probably identical to the factor that binds to the cAMP-responsive element of somatostatin. We have purified this factor, which is a protein of 43 kD in size.
Introns increase gene expression in cultured maize cells.
Authors: Callis J , Fromm M , Walbot V
Published: June 2007

Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adh1) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an 11.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adh1-S gene constructs lacking the 9 Adh1-S intervening sequences (introns) were expressed at levels 50- to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adh1-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adh1 gene was increased 100-fold by the addition of sequences containing the Adh1 intron 1. The Adh1 intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adh1 intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adh1 intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adh1 intron 1 and the intron from a second maize gene, Bronze1, stimulated expression from other promoters (cauliflower mosaic virus 35S and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adh1 and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.
Deletion analysis of the achaete-scute locus of Drosophila melanogaster.
Authors: Ruiz-Gómez M , Modolell J
Published: June 2007

The achaete-scute gene complex (AS-C) is involved in the development of the central and peripheral (sensory chaetae, sensilla) nervous system. To assess the contribution of the different parts of the complex in the generation of the adult chaetae pattern, we have determined the phenotypes and molecular positions of the breakpoints of 74 terminal deficiencies of the X chromosome. According to these and previous data, the AS-C is organized, distally to proximally, as follows: the achaete region, with most of its DNA (10 kb) located upstream from the putative achaete (T5) gene; an intermediate region, approximately 18 kb long, whose deletion only weakly affects the scute function; and the scute region, with most of the DNA critical for its function extending 4-5 kb upstream and 50 kb downstream of the putative scute (T4) gene. The DNA extending far upstream of the T5 gene and downstream of the T4 gene may provide chromatin conformations adequate for efficient expression of these genes. However, in the case of the T4 gene, the available data suggest the presence of a small number of elements, scattered in the long downstream region, that would respond to topological cues and cis-activate this gene in specific anatomical regions.
Molecular characterization of the zerknüllt region of the Antennapedia gene complex in Drosophila.
Authors: Rushlow C , Doyle H , Hoey T , Levine M
Published: June 2007

zerknüllt (zen) is unique among the 18 known homeo box genes in Drosophila since it is required for the differentiation of the dorsal-ventral pattern, and does not appear to be involved in the process of segmentation. Here we show that the zen region of the Antennapedia complex (ANT-C) consists of two closely linked homeo box genes, designated z1 and z2. The z1 and z2 transcription units show essentially identical patterns of expression during early development, which are consistent with the timing and sites of zen+ gene activity. The putative proteins encoded by z1 and z2 are highly divergent and are related only by virtue of homeo box homology. We have used P-element-mediated germ line transformation to show that z1 alone can provide zen+ gene function, suggesting that the z2 gene might be dispensable. The occurrence of closely linked homeo box genes that display similar patterns of expression is not unique to the zen locus. Such gene duplications might provide important clues to the evolution of the homeo box gene family in Drosophila and other organisms.
Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).
Authors: Lawlor E J , Baylis H A , Chater K F
Published: June 2007

In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.
Defects in lens fiber differentiation are linked to c-mos overexpression in transgenic mice.
Authors: Khillan J S , Oskarsson M K , Propst F , Kuwabara T , Vande Woude G F , Westphal H
Published: June 2007

We describe three strains of transgenic mice derived by embryo microinjection of DNA consisting of a long terminal repeat (LTR) of Moloney murine sarcoma virus (Mo-MSV) linked to the murine c-mos coding sequences. Southern analysis of the genomic DNA of these strains suggested that in each case the transgene had integrated at a different chromosomal location. The strains were characterized by dominant changes in secondary lens fiber differentiation. Shortly after birth, insufficient elongation of differentiating lens fibers and lack of basement membrane secretion resulted in breakdown of the posterior lens capsule. This, in turn led to posterior protrusion and swelling of lens tissue. In the course of the first 3 weeks after birth, globular lens cells began to fill the entire anterior and posterior chambers of the eye. Concomitantly, there was massive overexpression of c-mos RNA in the lens. Whereas this construct has high transforming activity when transfected into NIH-3T3 cells, no hyperplasia or neoplasia have been observed in the affected lenses. Increased expression of c-mos RNA was not confined to the lens of the eye but has been detected in any of several tissues tested.
Cell-type-specific expression of epidermal cytokeratin genes during gastrulation of Xenopus laevis.
Authors: Jamrich M , Sargent T D , Dawid I B
Published: June 2007

Analysis of the spatial pattern of expression of embryo-specific epidermal cytokeratin genes in Xenopus laevis shows earliest activity in the animal pole cells of stage-9 blastulae. These genes are transcribed predominantly in the epithelial or outer ectoderm, to a lesser extent in the sensorial or inner ectoderm, and at low levels if at all in other regions of the embryo. In the early gastrula the entire ectoderm, including preneural and preepidermal regions, expresses cytokeratin mRNAs; accumulation of these mRNAs in preneural cells is terminated after contact is made with involuting chordamesoderm. On the basis of this and earlier work (Sargent et al. 1986) we suggest that the pattern of expression of cytokeratin genes in frog embryogenesis is based on prelocalized components modulated by the inductive influence of involuting chordamesoderm. The cytokeratin proteins are deposited in the form of filamentous networks in both layers of the epidermis. In the epithelial layer, a much denser mesh of filaments is facing the outside of the embryo. This polarity is established at the onset of the polymerization of these filaments. Thus, intraembryonic and intracellular localization of keratin gene expression and protein deposition is established at the onset of activation of these genes.
Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli.
Authors: Grossman A D , Straus D B , Walter W A , Gross C A
Published: June 2007

The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response. We present experiments indicating a direct role for sigma 32 in controlling the heat shock response. Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32. Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32. These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.
Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.
Authors: Liao S M , Wu T H , Chiang C H , Susskind M M , McClure W R
Published: June 2007

The characterization in vitro of a newly discovered promoter (Psar) in the bacteriophage P22 immI region is described. Psar is located within the ant gene and is directed toward the major immI promoter, Pant. The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed. The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual. Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15- to 25-fold larger numbers than full-length transcripts. Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry. The effects of convergent transcription between Pant and Psar were investigated on linear and supercoiled templates. Active transcription from Pant interferes with full-length transcription from Psar; several factors that interfere with Pant initiation (e.g., Pant down-mutation, Mnt repressor protein, Arc repressor protein) result in indirect activation of sar RNA synthesis. The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex. The location and properties of Psar suggest an important regulatory function for sar RNA as a negative effector of ant expression. The results of Wu et al. (this issue) support this suggestion.
Control of gene expression in bacteriophage P22 by a small antisense RNA. II. Characterization of mutants defective in repression.
Authors: Wu T H , Liao S M , McClure W R , Susskind M M
Published: June 2007

Phage P22 produces antirepressor protein early after infection from a transcript initiated at the Pant promoter. After the first few minutes of infection, transcription from Pant is repressed by a protein encoded by the arc gene. Antirepressor is not produced late in infection, even though the antirepressor gene, ant, is transcribed from the late operon promoter Plate. We describe the isolation of P22 mutants that synthesize antirepressor from the Plate transcript. The mutations inactivate a promoter Psar, which lies within the ant coding sequence and directs the synthesis of sar RNA, a small antisense regulatory RNA complementary to the ant ribosome binding site. Characterization of the Psar down-mutants shows that transcription from Psar interferes with synthesis of antirepressor from both the Plate and Pant transcripts. Since sar RNA represses synthesis of antirepressor in trans, we propose that sar RNA base-pairs with ant mRNA to inhibit antirepressor synthesis at a post-transcriptional level. The role and importance of sar RNA in P22 biology are discussed.
Sequences in the pea rbcS-3A gene have homology to constitutive mammalian enhancers but function as negative regulatory elements.
Authors: Kuhlemeier C , Fluhr R , Green P J , Chua N H
Published: June 2007

The pea rbcS-3A gene, which codes for a key component of the photosynthetic machinery, requires light for its expression. Analysis of chimeric constructs in transgenic tobacco plants has shown that a 280-bp fragment from the 5' noncoding region can act as a light-inducible transcriptional enhancer. Further characterization of this enhancer identifies a 58-bp sequence containing two regulatory elements that can decrease transcription in the dark. One has a high degree of homology to the SV40 core enhancer, the other to the adenovirus 5 E1A enhancer and the constitutive part of the human interferon-beta gene enhancer. Deletion of the 58-bp sequence uncovers additional light-responsive elements (LREs) located further upstream and downstream.
Factors that interact with the rat albumin promoter are present both in hepatocytes and other cell types.
Authors: Babiss L E , Herbst R S , Bennett A L , Darnell J E
Published: June 2007

The rat albumin promoter inserted in adenovirus directs transcription in human and rodent hepatoma cells and in rodent hepatocytes (Friedman et al. 1986) and Babiss et al. (1986) but not in HeLa cells or myeloma cells. The nucleotides between -43 and -156 of the RNA start site of the rat albumin gene are required for this cell-specific expression. Protein binding studies (footprints, exonuclease III stops, and gel shifts) all indicate specific interaction in the -80 to -130 region of the gene with factors present in nuclear extracts of hepatocytes and hepatomas, but also from extracts of other cells that do not express the albumin gene. To observe albumin promoter binding, a smaller amount of extract of liver cell nuclei was required compared to extracts of HeLa cell or kidney cell nuclei. In addition, the various tests of DNA-protein interaction did not give qualitatively identical results with extracts from different cells. However, it seems clear that factors are present in several cell types where albumin genes are inactive that will bind to those DNA sequences demonstrated to be necessary for cell-specific expression of this gene. These factors could either be similar but nonidentical factors or the same factors that are modified differently in different cell types.
Reversibility of differentiation and proliferative capacity in avian myelomonocytic cells transformed by tsE26 leukemia virus.
Authors: Beug H , Blundell P A , Graf T
Published: June 2007

Chicken hematopoietic cells infected with E26 leukemia virus can be transformed into growth factor-dependent, rapidly proliferating cells that exhibit properties of immature myelomonocytic cells. Cells infected with a mutant of E26 that carries a temperature-sensitive lesion, presumably residing in the myb oncogene, differentiate into resting, macrophage-like cells when shifted from 37 degrees to 42 degrees C (Beug et al. 1984). Here we show that differentiated tsE26 cells gradually reacquire an immature phenotype and proliferative capacity when shifted back to 37 degrees C, provided that they are kept at 42 degrees C no longer than 4-8 days. We also show that DNA synthesis inhibitors do not prevent terminal differentiation at 42 degrees C but inhibit the complete reexpression of the immature phenotype in downshift experiments. Our results suggest that the reactivation of the E26 protein function can both induce a "retro-differentiation" and cell proliferation in myelomonocytic target cells.
Retroviruses and insertional mutagenesis in mice: proviral integration at the Mov 34 locus leads to early embryonic death.
Authors: Soriano P , Gridley T , Jaenisch R
Published: June 2007

Thirty-four transgenic mouse strains, each carrying a single proviral insert, were generated by infection of preimplantation and postimplantation embryos with retroviruses. Animals homozygous for proviral integrations were derived for all strains with the exception of Mov 24, where the provirus is inserted on the Y chromosome, and Mov 34. Embryos homozygous at the Mov 34 locus develop normally to the blastocyst stage and die shortly after implantation, indicating that virus integration resulted in a recessive lethal mutation. The provirus and flanking sequences were cloned and the virus was mapped to the 5' side of an abundantly and ubiquitously transcribed gene. Similar to the previously derived Mov 13 mutation, proviral integration at the Mov 34 locus interferes with the expression of the adjacent gene. These and our previous results indicate that of a total of 48 proviral integrations in the germ line, two resulted in transgenic mouse strains with recessive lethal mutations.
Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene.
Authors: Erickson J W , Vaughn V , Walter W A , Neidhardt F C , Gross C A
Published: June 2007

In Escherichia coli the product of the rpoH (htpR) gene, sigma 32, directs RNA polymerase to initiate transcription from heat shock promoters at all temperatures. Transcription of the heat shock genes is increased when cells are exposed to high temperatures because of increased transcription initiation by sigma 32-RNA polymerase. As a step toward understanding the regulation of the heat shock response we have examined the transcription of the rpoH gene. Using S1 mapping, promoter cloning, and in vitro transcription, we have identified the promoters and the terminator for the rpoH transcription unit. The rpoH transcripts are monocistronic and originate from at least three promoters. None of the promoters is recognized by sigma 32-RNA polymerase. Two are recognized by sigma 70-RNA polymerase and are active at both low and high growth temperatures. We do not know what form of RNA polymerase recognizes the third promoter. Transcripts from this promoter are abundant only at high temperature and are present after shift to the lethal temperature of 50 degrees C, even at times when there are no detectable transcripts from the other rpoH promoters. The amount of rpoH mRNA increases fivefold by 8 min after shift from 30 to 43.5 degrees C but rpoH mRNA synthesis increases by less than twofold, indicating that there is post-transcriptional control of the level of rpoH mRNA and presumably of sigma 32.
Transcript levels and translational control of hsp70 synthesis in Xenopus oocytes.
Authors: Horrell A , Shuttleworth J , Colman A
Published: June 2007

Until recently it was believed that Xenopus oocytes respond to heat shock by synthesizing the 70-kD heat shock protein hsp70 and that, uniquely amongst animal cell types, this response is mediated entirely at the translational level. This view has now been challenged and we present data that reevaluate the involvement of translational control in the heat shock response of Xenopus oocytes. RNase mapping shows that up to 13 pg of hsp70A and hsp70B mRNA are accumulated by fully grown oocytes in the absence of heat shock. These transcripts are retained stably during maturation, fertilization, early cleavage, and following heat shock. However, no hsp70 protein synthesis can be detected by two-dimensional polyacrylamide gel analysis of [35S]methionine-labeled proteins from completely defolliculated oocytes, either before or during heat shock. Oocytes injected with hsp70A DNA rapidly accumulate high levels of hsp70 mRNA in their cytoplasm at normal temperature. During heat shock these oocytes accumulate more transcripts, but they remain in the nucleus and cytoplasmic levels remain constant. Translation of hsp70 from these transcripts is readily detectable at non-heat shock and heat shock temperatures. We conclude that (1) "exogenous" hsp70 transcripts are efficiently translated and not masked at normal temperatures in oocytes, and (2) oocytes are able to selectively translate hsp70 mRNA during heat shock.
Expression of a homeo domain protein in noncontact-inhibited cultured cells and postmitotic neurons.
Authors: Odenwald W F , Taylor C F , Palmer-Hill F J , Friedrich V , Tani M , Lazzarini R A
Published: June 2007

The murine Hox 1.3 gene is one of six homeo box genes clustered on chromosome 6. Our analysis of Hox 1.3 cDNA and genomic clones indicates that the gene is organized into two exons and encodes a 270-amino-acid homeo domain protein. The predicted protein is rich in serine, glycine, and proline residues, and its homeo domain is identical to the Hox 2.1 domain. During embryogenesis, the gene is maximally expressed at midgestation but is also expressed to a lesser extent in many adult tissues possessing different cell lineages. Hox 1.3 transcripts are also present in cultured fibroblasts. The Hox 1.3 protein accumulates in the nuclei of nonconfluent cultured fibroblasts but is greatly diminished in contact-inhibited nongrowing cells. Thus, the expression of the Hox 1.3 gene correlates with growth in embryos and cultured cells. Paradoxically, it is also expressed in certain subsets of postmitotic, fully differentiated neurons, most notably the Purkinje neurons of the cerebellum, the pyramidal and dentate neurons of the hippocampus, and the motor neurons of the spinal cord. This complex pattern of expression suggests that Hox 1.3 may provide a function required by many cell types in addition to any role it may have in morphogenesis.
Regulation of the Drosophila dopa decarboxylase gene in neuronal and glial cells.
Authors: Beall C J , Hirsh J
Published: June 2007

A cis-regulatory element selectively required for the Drosophila melanogaster dopa decarboxylase gene (Ddc) in the central nervous system has been identified previously (Scholnick et al. 1986). Here, we show that at least one additional regulatory element is required for normal neuronal expression of Ddc. We find that Ddc is normally expressed in about 125 discrete neurons and in a diffused network comprising a subset of glial cells. The expression of in vitro-altered Ddc genes was studied by immunohistochemistry following germ line reintegration with P-element vectors. Normal neuron-specific Ddc gene expression requires both the initially identified element (element I) which is 60 bp upstream from the RNA start site, and an additional regulatory element located 800-2200 bp upstream. This latter element is required for neuronal expression but is not necessary for glial expression of Ddc. We provide a model to explain how interactions between multiple regulatory elements may serve to specify cell-specific gene expression.
A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation.
Authors: Lamond A I , Konarska M M , Sharp P A
Published: June 2007

We have analyzed the pathway of mammalian spliceosome assembly in vitro using a mobility retardation assay. The binding of splicing complexes to both wild-type and mutant beta-globin pre-RNAs was studied. Three kinetically related, ATP-dependent complexes, alpha, beta, and gamma, were resolved with a wild-type beta-globin substrate. These complexes formed, both temporally and in order of decreasing mobility, alpha----beta----gamma. All three complexes contained U2 snRNA. The RNA intermediates of splicing, i.e., free 5' exon and intron lariat + 3' exon, were found predominantly in the gamma complex. The RNA products of splicing, i.e., ligated exons and fully excised intron lariat, were found in separate, postsplicing complexes which appeared to form via breakdown of gamma. Mutations of the 5' splice site, which caused an accumulation of splicing intermediates, also resulted in accumulation of the gamma complex. Mutations of the 3' splice site, which severely inhibited splicing, reduced the efficiency and altered the pattern of complex formation. Surprisingly, the analysis of double mutants, with sequence alterations at both the 5' and 3' splice sites, revealed that the 5' splice site genotype was important for the efficient formation of a U2 snRNA-containing alpha complex at the 3' splice site. Thus, it appears that a collaborative interaction between the separate 5' and 3' splice sites promotes spliceosome assembly.
The mouse eye lens obsolescence (Elo) mutant: studies on crystallin gene expression and linkage analysis between the mutant locus and the gamma-crystallin genes.
Authors: Quinlan P , Oda S , Breitman M L , Tsui L C
Published: June 2007

Previous studies showed that the mouse Elo (eye lens obsolescence) mutation is located on chromosome 1, at a site near the Len-1 locus, which is defined by a set of polymorphic gamma-crystallin proteins. To investigate further the relationship between Elo and the gamma-crystallins, we have examined the steady-state levels of gamma-crystallin transcripts in normal and mutant eyes and analyzed the linkage relationship between the Elo locus and the gamma-crystallin genes. Our data showed that, while gamma-crystallin mRNA levels are preferentially reduced in the mutant eyes, the mutation does not seem to map within the gamma-crystallin gene cluster. The distance between Elo and the gamma 6 gene (the most proximal gamma-crystallin gene member) is estimated to be 1.4 +/- 0.9 cM, whereas that between gamma 6 and the distantly linked gamma 2 gene is 2.7 +/- 1.3 cM. Our data also suggest the possibility of recombination hot spots with the gamma-crystallin gene cluster.
Identification of gene products expressed in the developing chick visual system: characterization of a middle-molecular-weight neurofilament cDNA.
Authors: Zopf D , Hermans-Borgmeyer I , Gundelfinger E D , Betz H
Published: June 2007

A cDNA library enriched for transcripts that accumulate from embryonic day 7 until day 1 posthatching was constructed from poly(A)+ RNA of chick optic lobe tissue. From this library, three recombinants were isolated that correspond to neural mRNAs appearing during the major period of synaptogenesis in the retinotectal system. One of these recombinants (OZ 11) was identified as a middle-molecular weight neurofilament (NF-M) cDNA which, together with two overlapping clones, encodes part of the rod and the entire tail region of NF-M. In situ hybridization revealed NF-M mRNA to be highly expressed in regions of the chick central nervous system, which contain multipolar and/or long-projection neurons.
Distinct factors with Sp1 and NF-A specificities bind to adjacent functional elements of the human U2 snRNA gene enhancer.
Authors: Ares M , Chung J S , Giglio L , Weiner A M
Published: June 2007

The enhancer regions of mammalian and avian U1 and U2 small nuclear RNA (snRNA) genes are unusual in containing the sequence GGGCGG (GC-box) immediately upstream from the sequence ATGCAAAT (octamer). We made point mutations in the human U2 snRNA enhancer and tested them for the ability to direct U2 transcription in HeLa cells, as well as for the ability to form complexes with factors present in HeLa cell nuclear extracts. We show that neither the GC-box nor the octamer alone is sufficient for enhancer activity in vivo. Mutations in the GC-box reduce the ability of enhancer DNA fragments to bind a factor (probably Sp1), whereas mutations in the octamer independently reduce the ability to bind a second factor (probably nuclear factor A, NF-A). The results suggest that adjacent binding of Sp1 and NF-A is an important feature of some U-snRNA gene enhancers.
Lens-specific enhancer in the third intron regulates expression of the chicken delta 1-crystallin gene.
Authors: Hayashi S , Goto K , Okada T S , Kondoh H
Published: June 2007

We have previously shown that the tissue specificity determinant of the chicken delta 1-crystallin gene lies 3' of position -100 (Hayashi et al. 1985). Since the promoter of the gene (delta 1-crystallin promoter) did not show any tissue specificity, we examined various segments of the delta 1-crystallin gene for a tissue-specific enhancer activity by placing each segment downstream of a heterologous transcriptional unit coding for chloramphenicol acetyltransferase (CAT) and by transfecting chicken tissues in primary culture. We found that a segment spanning the third intron bears a strong lens-specific enhancer activity. This "delta 1-crystallin enhancer" activates transcription from the delta 1-crystallin promoter 20- to 40-fold in lens cells and to various degrees with other promoters. Deletion analysis of the enhancer region indicated that it covered nearly 1 kb but did not indicate clear-cut boundaries. For its enhancer effect the core region of 120 bp and associations with certain adjoining regions were required. Removal of the enhancer from the gene totally abolished delta 1-crystallin expression, and reinsertion of the enhancer in either upstream, internal, or downstream positions restored expression. We conclude that the delta 1-crystallin enhancer is an essential and major determinant for lens-specificity of delta 1-crystallin expression.
Closely linked DNA elements control the expression of the Sgs-5 glue protein gene in Drosophila.
Authors: Shore E M , Guild G M
Published: June 2007

cis-acting sequence regions involved in the regulation of Sgs-5 gene expression were mapped by testing DNA segments containing the Sgs-5 RNA coding region and various amounts of adjacent sequences for the ability to express Sgs-5 RNA. Following injection of the DNA segments into Drosophila embryos, expression of the gene was assayed in the salivary glands of the injected animals after they developed to third instar larvae, these somatically transformed individuals serving as an in vivo transient expression system. The information necessary for the expression of Sgs-5 is contained within 109 bp upstream and 69 bp downstream of the transcribed region. Somatic transformation experiments also show that some feature within the limits of a 1012-bp DNA segment containing the Sgs-5 RNA coding region derived from the Sgs-5 RNA null stock CA-2 must be responsible for the lack of transcription from this allele. The only DNA sequence differences between active and null alleles, within the 1012 bp, are seven single-base-pair substitutions between -84 bp and +175 bp relative to the RNA start site. One or a combination of these sites are likely contributors to the transcriptional inactivity of the Sgs-5CA2 allele.
Specific pattern of gene expression during induction of mouse erythroleukemia cells.
Authors: Fraser P J , Curtis P J
Published: June 2007

We have studied the expression of several characterized genes during induction of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide (DMSO) and have observed a specific pattern of changes in transcriptional activity and steady-state RNA levels associated with erythroid differentiation. During induction there is a gradual, steady decrease in total transcriptional activity and RNA content per cell, which by day 3 of DMSO treatment amounts to less than 50% of the level in the uninduced cell. During this time we observe increases in transcriptional activity for 5-aminolevulinic acid synthase, carbonic anhydrase form II, and band 3 coordinate with the large increase in beta-globin gene transcription. The results also demonstrate an early decrease in transcription for carbonic anhydrase form I, which precedes decreases in transcription for glyceraldehyde phosphate dehydrogenase and rRNA genes. Changes in steady-state RNA levels reflected changes in transcriptional activity during induction except for carbonic anhydrase II mRNA. These results represent the first report characterizing the regulated expression at transcriptional and posttranscriptional levels of several known genes that are characteristically expressed in the erythrocyte. The results demonstrate that coordinate gene expression in erythroid differentiation occurs primarily at the level of transcription.
Dorsal and neural expression of a tyrosine kinase-related Drosophila gene during embryonic development.
Authors: Haller J , Côté S , Brönner G , Jäckle H
Published: June 2007

Sequence analysis of an embryonic transcript of Drosophila predicts a tyrosine protein kinase-related gene. The prediction is based on several protein domains that are homologous to the functional domains of kinase-related oncogenes and several serine, threonine, and tyrosine protein kinases. For this reason, we named this gene Drosophila tyrosine kinase related (dTKR). dTKR maps into chromosome band 2R 60F1. It is initially expressed at blastoderm stage, showing transient transcript accumulations at dorso-lateral positions of the embryo and differences along its longitudinal axis. At later stages of embryogenesis, dTKR transcripts are found exclusively in neural anlagen. Both the region-specific pattern of expression and the putative kinase function are consistent with the suggestion of a regulatory role for this gene during development, which remains to be elucidated.
The decapentaplegic gene is required for dorsal-ventral patterning of the Drosophila embryo.
Authors: Irish V F , Gelbart W M
Published: June 2007

The decapentaplegic gene (dpp), which encodes a growth factor-like protein (Padgett et al. 1987), is implicated in several morphogenetic events in Drosophila melanogaster. We define here a novel embryonic function encoded by dppHin+ alleles of the dpp gene. dppHin null homozygotes die as ventralized embryos. dppHin activity is not required in the maternal germ line since lack of dppHin function during oogenesis has no effect on the zygotic phenotype. Since dppHin null embryos are already abnormal early in gastrulation, the dppHin product is an early-acting, strictly zygotic function involved in establishing the embryonic dorsal-ventral pattern. Several maternally acting dorsalizing genes are thought to be required for the establishment of a dorsal-ventral morphogenetic gradient (Anderson et al. 1985b). We have examined the interactions of dppHin mutations with three of these genes. Embryos null for dppHin and derived from a mother homozygous for a dorsalizing mutation exhibit a lateralized phenotype, indicating that the dorsal-ventral identity of the epidermis in part derives from the direct or indirect regulation of dppHin activity by these genes.
OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression through an RNase III-dependent mechanism.
Authors: Krinke L , Wulff D L
Published: June 2007

OOP RNA is a major short (77 bases) transcript that is made from bacteriophage lambda DNA both in vivo and in vitro. OOP RNA is synthesized in the opposite direction to mRNA for the lambda cII gene, and the final 55 bp of the OOP region overlaps the 3' end of the cII gene. We find that a multicopy plasmid containing an OOP DNA fragment inhibits cII expression from a derepressed prophage by approximately 100-fold, using an in vivo assay in which cII protein activates galactokinase synthesis from a cII-dependent promoter on a multicopy plasmid. A large inhibitory effect is also observed when the po promoter for OOP RNA is replaced by the strong lambda pL promoter, but not when po is deleted. Plasmids that provide a large excess of "anti-OOP" RNA (RNA that is complementary to OOP RNA) make OOP RNA a less effective inhibitor of cII expression. Inhibition by the OOP DNA plasmid is not observed in an Escherichia coli strain deficient in RNase III. We propose that the 3' end of cII mRNA and OOP RNA form a double-stranded complex that is a substrate for the host enzyme RNase III, resulting in degradation of cII mRNA. Deletion studies on the OOP DNA plasmid indicate that no specific sequence between the promoter and terminator stem structure is required for the inhibitory effect. Lambda cII expression from an induced prophage is increased twofold in the presence of a large excess of anti-OOP RNA. This experiment, in which the prophage is the sole source of OOP RNA, suggests a physiological role for OOP RNA in regulating cII-gene expression.
Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae.
Authors: Nikawa J , Cameron S , Toda T , Ferguson K M , Wigler M
Published: June 2007

We have isolated and characterized normal and mutant alleles of many of the genes of the RAS/adenylyl cyclase pathway of the yeast Saccharomyces cerevisiae. Manipulation of those genes has revealed a system for feedback control that can modulate cAMP levels over at least a 10,000-fold range. The feedback control depends upon the activity of the cAMP-dependent protein kinases and requires the presence of the CDC25 and RAS proteins. The capacity for such dramatic control of cAMP levels raises fundamental questions about the normal mechanism of action of the cAMP signaling system in yeast.
A 3' enhancer contributes to the stage-specific expression of the human beta-globin gene.
Authors: Trudel M , Costantini F
Published: June 2007

The human beta-globin and G gamma-globin genes are expressed at different stages of human development and also show distinct temporal patterns of expression when transferred into the mouse germ line. In transgenic mice, the beta-globin gene is expressed only in fetal and adult erythroid cells, whereas the G gamma-globin gene is active only in embryonic erythroid cells. Previous experiments suggested that beta-globin 3' sequences were important for expression in mouse fetal and adult erythroid cells, and in this paper we directly demonstrate the presence of an enhancer in the 3'-flanking region of the gene. First, deletion of sequences between 605 and 895 bp, 3' to the poly(A) site, results in a 10-fold reduction in the average level of expression of the beta-globin gene in transgenic mouse fetal livers. Second, a DNA fragment including beta-globin 3'-flanking sequences [425-1480 bp from the poly(A) site], in either orientation, activates transcription from the otherwise silent G gamma-globin promoter in the mouse fetal liver; DNA sequences between 150 and 730 bp or between 920 and 1680 bp, 3' to the beta-globin gene, are inactive by this assay. Together, these experiments identify an enhancer, in the region approximately 600-900 bp, 3' to the beta-globin poly(A) site, which contributes to the differential stage-specific expression of the beta-globin and G gamma-globin genes.