bluuebunny/crossref_metadata_2025 huggingface dataset:
There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology
Authors: Hawley R. Scott , Irick Holly , Haddox Deana A. , Whitley Michelle D. , Arbel Tamar , Jang Janet , McKim Kim , Zitron Anne E. , New Christine , Childs Geoffrey , Lohe Allan
Published: May 2005

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology‐dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene‐diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co‐orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology‐independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle. © 1993 Wiley‐Liss, Inc.
The independent stage‐specific expression of the 18‐kDa heat shock protein genes during microsporogenesis in Zea mays L
Authors: Atkinson Burr G. , Raizada Manish , Bouchard Robert A. , Frappier J. Roger H. , Walden David B.
Published: May 2005

The small (18‐kDa) heat shock proteins (hsps) of maize are encoded by a complex multigene family. In a previous report, we described the genetic information from cDNAs encoding two different members of the family. In this communication, we report the isolation and characterization of cDNA and genomic clones encoding information for a third member of this hsp family (c/gMHSP18–1). DNA fragments containing nucleotide sequences common to, or specific for, each of these characterized 18‐kDa genes were prepared and used as probes to assess the expression of these genes during microsporogenesis and development of the gametophyte in an inbred line of maize (Oh43). Our results demonstrate (1) that mRNA transcripts encoding the 18‐kDa hsps are expressed and/or accumulate during microsporogenesis, and (2) that genes encoding two of the characterized 18‐kDa hsps are expressed and/or accumulate independently, in a stage‐specific manner during microsporogenesis. These observations imply that the stage‐specific expression of particular 18‐kDa hsp genes results from gene‐specific regulation during microsporogenesis and gametophyte development rather than from an overall activation of the heat shock or stress response. © 1993Wiley‐Liss, Inc.
Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos
Authors: Ali Adnan , Krone Patrick H. , Heikkila John J.
Published: May 2005

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT‐PCR, we found that this gene was first heat‐inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′‐flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′‐flanking region. RT‐PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′‐ and 3′‐flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat‐inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis‐acting DNA sequences required for correct heat‐inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley‐Liss, Inc.
The induction of pyruvate kinase synthesis by heat shock in Xenopus laevis embryos
Authors: Marsden M. , Nickells R. W. , Kapoor M. , Browder L. W.
Published: May 2005

Heat‐shocked Xenopus embryos have an unusually complex heat shock response. The dominant heat shock protein (Hsp) has a relative molecular mass (Mr) of 62,000 D (Hsp62). Affinity‐purified IgGs against the glycolytic enzyme pyruvate kinase (PK; EC 2.7.1.40) specifically immunoprecipitated Hsp62 from extracts of embryos that had been heat‐shocked at 37°C for 30 min. Thus, Hsp62 and pyruvate kinase are immunologically cross‐reacting. Electrophoretic separation of PK isoforms suggests that heat‐shocked Xenopus embryos increase synthesis of an isoform of PK. Thermal denaturation studies suggest that this isoform has enhanced thermal stability. The identification of PK as an Hsp is discussed within the context of a physiological requirement for elevated levels of anaerobic glycolysis in heatstressed cells as a vital component of the acquisition of thermotolerance. © 1993Wiley‐Liss, Inc.
Activity of a microinjected inducible murine hsp68 gene promoter depends on plasmid configuration and the presence of heat shock elements in mouse dictyate oocytes but not in two‐cell embryos
Authors: Bevilacqua Arturo , Mangia Franco
Published: May 2005

After fertilization in the mouse, the zygotic genome is activated in two‐cell embryos by the spontaneous expression, among other genes, of the major inducible heat shock gene, hsp68, in the absence of heat‐inducibility of heat shock genes. To obtain information on this phenomenon, we have probed one‐ and two‐cell embryo's ability to express microinjected reporter DNA constructs, containing the Escherichia coli lacZ gene driven by promoters from early SV40 genes, the human ß‐actin gene, and the normal or HSE‐deleted mouse hsp68 gene. Activity of these promoters was also tested in mouse granulosa cells and dictyate oocytes, as a function of circular/linear construct configuration and occurrence of heat shock. The hsp68 promoter was heat‐inducible in both granulosa cells and oocytes. Its heat activation required the presence of HSEs and, in the oocytes, of construct linear configuration. In the embryos however, this promoter was expressed in dependently of the presence of HSEs and of construct configuration, and its activity was not affected by heat shock. When constructs with early SV40 and ß‐actin promoters were injected into one‐cell embryos, they appeared to be inactivated with the first embryonic cleavage, in agreement with previous observations [Wiekowski et ai., 1992]. By contrast, both normal and HSE‐deleted hsp68 promoters maintained their activity through the first cleavage, providing the first evidence of a gene escaping such transcriptional repression. Present results confirm previous findings on hsp68 expression during early mouse development, and suggest that this activation is mediated by a factor(s) other than HSF. © 1993Wiley‐Liss, Inc.
Development and tissue‐specific distribution of mouse small heat shock protein hsp25
Authors: Gernold M. , Knauf U. , Gaestel M. , Stahl J. , Kloetzel P.‐M.
Published: May 2005

We have investigated the developmental and tissue‐specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13–20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related α, B‐crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism. © 1993Wiley‐Liss, Inc.
Lack of concordance between heat shock proteins and the development of tolerance to teratogen‐induced neural tube defects
Authors: Finnell Richard H. , Van Waes Michael , Bennett Gregory D. , Eberwine James H.
Published: May 2005

The present study was undertaken to examine the role of heat shock response in the development of tolerance and cross‐tolerance in an in vivo murine model of teratogen‐induced neural tube defects. The experimental paradigm designed to address this question was to utilize inbred mouse strains that differed in their sensitivity to hyperthermia and valproic acid induced neural tube defects, subjecting the dams to subteratogenic pretreatments with either heat or valproic acid at two different timepoints during development prior to the administration of the teratogenic insult. A statistically significant reduction in the frequency of neural tube defects and/or embryolethality following a pretreatment in dams subsequently exposed to a teratogenic treatment was considered evidence for the induction of tolerance. This was observed in the SWV embryos exposed to the 38°C pretreatment at 8:06 and to embryos exposed to either pretreatment temperature at 8:10 priorto a teratogenic heat shock at 8:12. In the LM/Bc embryos, only the 41°C pretreatment at 8:06 induced thermotolerance. There was no evidence of tolerance induced in either mouse strain using valproic acid. On the other hand, cross‐tolerance was clearly demonstrated in this study, with a low temperature (41°C) pretreatment successfully protecting SWV fetuses from a subsequent teratogenic treatment with valproic acid, while valproic acid (200 mg/kg) was effective in reducing the risk of hyperthermia‐induced neural tube defects in the LM/Bc fetuses. In all instances, tolerance was induced in the absence of significant induction of hsp synthesis. The lack ofconcordance between hsps and thermotolerance suggests that some other factor(s) is involved in conferring thermotolerance on developing murine embryos. © 1993 Wiley‐Liss, Inc.
Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies, antisense RNA, and deoxyoligonucleotides
Authors: Brice Eleanor C. , Wu Jie‐Xin , Muraro Raffaella , Adamson Eileen D. , Wiley Lynn M.
Published: May 2005

Two‐cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand‐receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation. © 1993Wiley‐Liss, Inc.
Possible autocrine/paracrine actions of insulin‐like growth factors during embryonic development: Expression and action of IGFs in undifferentiated P19 cells
Authors: Eicher Dorothea Jenkins , Moats‐Staats Billie M. , Stiles Alan D. , D'Ercole A. Joseph
Published: May 2005

The insulin‐like growth factors I and II (IGF I and II) and their cell surface receptors are expressed in the mammalian embryo and may function as autocrine or paracrine growth factors during early development. P19 embryonic carcinoma cells, derived from a 7.5 day mouse embryo, were used as a model for a functional study of the IGF system in post‐implantation embryogenesis. Undifferentiated P19 cells synthesized IGF I and II, the type I and II IGF receptors, and IGF binding proteins (IGF BP2, IGF BP3, and IGF BP4). P19 cells showed an increase in thymidine incorporation of 150% of control with a 4 hour incubation of IGF I (10 ng/ml) or IGF II (100 ng/ml) and an increase in cell viability compared to control cells during 24 hours of serum starvation. In both experiments IGF I was more potent than IGF II. Endogenous concentrations of IGF I and II in conditioned media were low compared to the doses of exogenous IGFs required for biologic effect, but nonetheless contributed significantly to baseline DNA synthesis, as demonstrated by inhibition of IGF actions with specific antibodies. Cell surface associated IGF BPs bound more radiolabeled IGF than IGF receptors, as determined by binding studies and affinity cross‐linking. IGF I and IGF II appeared to regulate production of IGF BP2, suggesting that the IGFs may regulate their own actions by altering the abundance of their binding proteins. © 1993Wiley‐Liss, Inc.
Rapid induction and clearance of TGFβ1 is an early response to wounding in the mouse embryo
Authors: Martin Paul , Dickson Marion C. , Millan Fergus A. , Akhurst Rosemary J.
Published: May 2005

The TGFβ family of growth factors has been implicated as playing a significant role in many aspects of embryonic morphogenesis, and also as a mediator of adult tissue repair processes. Unlike the situation in the adult, rissue repair in the embryo does not result in scarring, and it has been suggested that this might be due, in part, to reduced levels of growth factors, particularly TGFβ, at the wound site. We have examined the expression patterns of TGFβ genes following wounding of limb bud lesions in cultured Ell.5 mouse embryos. The timetable of wound closure was investigated by standard light and electron microscopy from the time of wounding until the lesion had re‐epithelialised 24 hours later. The expression of transcripts for each of the three TGFβ genes was examined at various time points during the healing process using radioactive in situ hybridisation to tissue sections and wholemount non‐radioactive in situ hybridisation to embryo pieces. Within l to 3 hours of wounding, transcripts encoding TGFβl were rapidly induced within the epithelial cells of the wound margin, particularly those cells at the ventral aspect of the wound. By 3 to 6 hours post‐wounding, TGFβl transcripts were detectable in the mesenchyme of the wound bed. No TGFβS induction was observed, and possible TGFβ2 induction was largely obscured by endogenous expression associated with pre‐cartilage mesenchymal condensation. Immunocytochemical analysis of tissue sections of the wound demonstrated a rapid induction of TGFβl protein within l hour post‐wounding, but also a subsequent rapid clearance of the protein from the wound site such that, by 18 hours post‐wounding, TGFβl levels had returned to near background. These data are discussed in terms of the molecular mechanisms underlying embryonic wound healing and the significance of the results to an understanding of scarring following adult tissue repair. © 1993Wiley‐Liss, Inc.
Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process
Authors: Theologis Athanasios , Oeller Paul W. , Wong Lu‐min , Rottmann William H. , Gantz David M.
Published: May 2005

Fruit ripening is one of the most dramatic developmental transitions associated with extensive alteration in gene expression. The plant hormone ethylene is considered to be the causative ripening agent. Transgenic tomato plants were constructed expressing antisense or sense RNA to the key enzyme in the ethylene (C2H4) biosynthetic pathway, 1 ‐aminocyclopropane‐1‐carboxylate (ACC) synthase using the constitutive CaMV 35S and fruit specific E8 promoters. Fruits expressing antisense LE‐ACS2 RNA produce less ethylene and fail to ripen only when ethylene production is suppressed by more than 99% (>0.1 nl/g fresh weight). Ethylene production is considerably inhibited (50%) in fruits expressing sense LE‐ACS2 RNA. Antisense fruits accumulate normal levels of polygalacturonase (PG), ACC oxidase (pTOM13), E8, E17, J49, and phytoene desaturase (D2) mRNAs which were previously thought to be ethylene‐inducible. E4 gene expression is inhibited in antisense fruits and its expression is not restored by treatment with exogenous propylene (C3H6). Antisense fruits accumulate PG mRNA, but it is not translated. Immunoblotting experiments indicate that the PG protein is not expressed in antisense fruits but its accumulation is restored by propylene (C3H6) treatment. The results suggest that at least two signal‐transduction pathways are operating during tomato fruit ripening. The independent (developmental) pathway is responsible for the transcriptioncl activation of genes such as PG, ACC oxidase, E8, E17, D2, and J49. The ethylene‐dependent pathway is responsible for the transcriptional and posttranscriptional regulation of genes involved in lycopene, aroma biosynthesis, and the translatability of developmentally regulated genes such as PG. © 1993Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.

© 1993Wiley‐Liss, Inc.
Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons
Authors: Yu C. , Brussaard A. B. , Yang X. , Listerud M. , Role L. W.
Published: May 2005

Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide‐mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations ≥ 20 μM. This temperature sensitive, saturable component is effectively competed by single nucleotides of ATP and AMP and is reminiscent of receptor‐mediated endocytosis of oligonucleotides described in non‐neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro.

Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit α3; nAChR α3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by =80%–90%. Furthermore, following antisense deletion of α3, “mutant” nAChRs with distinct functional characteristics are expressed.

In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons. In addition, the functional studies emphasize the need for sensitive and specific assay following antisense deletion, since other homologous gene products may substitute for the targeted gene resulting in new phenotypes that are subtly different from wild type. © 1993Wiley‐Liss, Inc.
Opposite functions of Jun‐B and c‐jun in growth regulation and neuronal differentiation
Authors: Schlingensiepen Karl‐Hermann , Schlingensiepen Reimar , Kunst Mechthild , Klinger Ingrid , Gerdes Wilhelm , Seifert Wilfried , Brysch Wolfgang
Published: May 2005

Induction of the jun‐B and/or c‐jun transcription factors is part of the immediate early response to diverse stimuli that induce alterations in cellular programs. While c‐jun is a protooncogene whose expression is required for induction of cell proliferation, jun‐B has recently been found to be induced by stimuli inducing differentiation in various cell lines. Furthermore, its expression is largely restricted to differentiating cells during embryogenesis. To determine the functional significance of these findings, we used antisense phosphorothioate oligodeoxynucleotides to inhibit expression of the two genes in proliferating and neuronally differentiating cells. While selective inhibition of c‐jun expression reduced proliferation rates, inhibition of jun‐B protein synthesis markedly increased proliferation in 3T3 fibroblasts, human mammary carcinoma cells and PC‐12 pheochromocytoma cells, suggesting jun‐B involvement in negative growth control. Neuronal differentiation of PC‐12 cells induced by nerve growth factor (NGF) was prevented by inhibition of jun‐B protein synthesis. PC‐12 cells not only failed to grow neurites but also remained in the proliferative state. Furthermore, in cultured primary neurons from rat hippocampus, inhibition of jun‐B expression, again, markedly reduced morphological differentiation. Conversely, inhibition of c‐jun protein synthesis enhanced morphological differentiation of both primary neurons and PC‐12 tumor cells. Thus, jun‐B expression is required for neuronal differentiation and its balance with c‐jun activity is involved in regulating key steps in proliferation and differentiation processes. © 1993Wiley‐Liss, Inc.
Juvenile hormone action to suppress gene transcription and influence message stability
Authors: Jones Grace , Venkataraman Venkateswar , Manczak Maria , Schelling Douglas
Published: May 2005

Proteins normally expressed in high abundance only at larval‐pupal metamorphosis in Trichoplusia ni were examined in a comparative analysis of the role and level of hormonal control of their expression. Some related proteins in the hemocyanin‐superfamily (i.e., an acidic protein [AJHSP1] and two basic proteins [BJHSP1, BJHSP2]) were shown by nuclear run‐on analysis to be specifically transcriptionally suppressed by juvenile hormone (JH), while transcription of another member of that family which is also metamorphosis‐associated (arylphorin) was not specifically sensitive to JH. The stability of the mRNA for those members transcriptionally down‐regulated by JH appeared to decrease under high JH conditions. While each protein was resorbed to some extent by the prepupal fat body, only the two basic proteins were quantitatively cleared from prepupal hemolymph. The JH‐sensitive proteins studied appear to be encoded in single copy genes not immediately juxtaposed in the genome. These and previous studies now permit a more comprehensive understanding of the different combinations of mechanisms involving transcription, mRNA stability, translation, and protein clearance that operate to regulate these metamorphosis‐associated proteins. © 1993Wiley‐Liss, Inc.
A murine CDC25/ras‐GRF‐related protein implicated in ras regulation
Authors: Chen Luping , Zhang Li‐jia , Greer Peter , Tung Pierre S. , Moran Michael F.
Published: May 2005

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release‐inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras‐GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9‐kb GRF2 messenger RNA was highly expressed in brain, but GRF2‐specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely‐expressed protein that is highly related to CDC25Mm/ras‐GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras‐GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras‐dependent proliferation and differentiation in early mouse development. © 1993Wiley‐Liss, Inc.
Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras‐related gene rap1
Authors: Rebstein P. J. , Weeks G. , Spiegelman G. B.
Published: May 2005

The rap1 gene of Dictyostelium discoideum is a member of the ras‐gene superfamily of low molecular weight GTPase proteins. The rapl gene is expressed both during growth and development in D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F‐actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rapl transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rapl transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rapl transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rapl transformant. We propose that the Rapl protein functions in the regulation of cell morphology in D. discoideum. © 1993Wiley‐Liss, Inc.
Developmental requirements for the ecdysoneless (ecd) locus in Drosophila melanogaster
Authors: Henrich Vincent C. , Livingston Leon , Gilbert Lawrence I.
Published: May 2005

The ecdysoneless locus in Drosophila melanogaster has been defined previously by a single conditional mutation, I(3)ecd1, that causes an ecdysteroid deficit and larval death at the restrictive temperature, 29°C, although the primary role of the mutation in developmental processes has been unclear. Gene dosage and complementation studies reported here for ecd1 and five nonconditional lethal alleles indicate that the ecd locus plays prezygotic and postzygotic roles essential for normal embryonic development, the successful completion of each larval molt, adult eclosion, and female fertility. The ecd locus is also required for normal macrochaete differentiation. For each observed phenotype, the severity of mutational effects was correlated with ecd mutant genotypes. In all cases, ecd1 homozygotes were least affected. Mutants heteroallelic for ecd1 and any one of four nonconditional recessive mutations were more severely affected than ecd1 homozy‐gotes, revealing these as hypomorphic alleles. For all phenotypic effects, mutants heteroallelic for ecd1 and a dominant mutation (ecd3D) were most severely affected. These individuals died during embryogenesis at 29°C and developed no macrochaetes on the dorsal thorax when transferred to 29°C during the white prepupal stage. The ecd3D mutation also caused female semisterility in heterozygotes. Ecdysteroid regulation has been implicated previously in all the developmental processes disrupted by these ecd mutations except for macrochaete differentiation. © 1993 Wiley‐Liss, Inc.
Analysis of the subtelomeric regions of macronuclear gene‐sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?
Authors: Maercker Christian , Lipps Hans Joachim
Published: May 2005

The subtelomeric regions of macronuclear gene‐sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene‐sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley‐Liss, Inc.
Antisense inhibition of nuclear‐encoded cytochrome C oxidase subunits IV and VIIc activity in the pre‐implantation embryo
Authors: Ao Asangla , Erickson Robert P. , Rosenthal Nancy H. , Grossman Lawrence I.
Published: May 2005

It had not previously been known whether synthesis of nuclear‐encoded mitochondrial subunits occurs in pre‐implantation embryos. We have used cytoplasmic injections of antisense RNA transcribed in vitro to study this question. Capped, in vitro transcribed RNA antisense to either cytochrome coxidase subunit IV or VIIc injected into each cell at the two‐cell stage markedly inhibited synthesis of adenine nucleotides by the 8‐ to 16‐cell stage, whereas injection of the cognate sense RNAs gave levels similar to those previously published for normal embryos. These results strongly suggest that translation of nuclear‐encoded mRNAs for mitochondrial subunits is required during pre‐implantation development. It was of additional interest that, not only was ATP decreased, but ADP and AMP as well, with the effect that the charge ratio remained constant. The results also suggest, therefore, that the mechanism by which cells normally regulate their charge ratio, thought to be with adenylate deaminase, is already in place. © 1993Wiley‐Liss, Inc.
Sea urchin maternal mRNA classes with distinct developmental regulation
Authors: Kelso‐Winemiller Leslie , Yoon Joonwon , Peeler Margaret T. , Winkler Matthew M.
Published: May 2005

Previous studies of newly synthesized proteins during early development in sea urchins have revealed several different patterns of synthesis that can be used to predict the existence of mRNA classes with distinct regulatory controls. We have identified clones for abundant maternal mRNAs that are actively translated during early development by screening a cDNA library prepared from polysomal poly(A) + RNA isolated from 2‐cell stage (2‐hour) Strongylocentrotus purpuratus embryos. Probes prepared from these cDNA clones and several previously characterized maternal mRNA cDNAs were used to compare relative levels of individual mRNAs in eggs and embryos and their translational status at various developmental stages. These abundant mRNAs can be classified into two major groups which we have termed cleavage stage‐specific (CSS) and post cleavage stage (PCS) mRNAs. The relative levels of the CSS mRNAs are highest during the rapid cleavage stage and decrease dramatically at the blastula stage (12‐hours). In contrast, PCS mRNAs are present at relatively low levels during the rapid cleavage stage and then increase at the blastula stage. Polysome partition profiles reveal that CSS mRNAs are translated more efficiently than PCS mRNAs in the unfertilized egg, at fertilization, and during the cleavage stages. Following the blastula stage, some CSS transcripts move out of polysomes and accumulate as untranslated RNAs, while newly transcribed PCS mRNAS are recruited into polysomes. These data suggest that the rapid cell cycles following fertilization require high levels of specific cleavage stage proteins, and the synthesis of these proteins occurs preferentially over PCS mRNAs. © 1993Wiley‐Liss, Inc.
Gene regulation in Drosophila spermatogenesis: Analysis of protein binding at the translational control element TCE
Authors: Kempe Elisabeth , Muhs Beate , Schäfer Mireille
Published: May 2005

We have previously identified a 12 nucleotide long sequence element, the TCE, that was demonstrated to be necessary for translational control of expression in the male germ line of Drosophila melanogaster (Schäfer et al., 1990). It is conserved among all seven members of the Mst(3)CGP gene family, that encode structural proteins of the sperm tail. The TCE is invariably located in the 5′ untranslated region (UTR) at position + 28 relative to the transcription start site. In this paper we analyse the mode of action of this element. We show that protein binding occurs at the TCE after incubation with lestis protein extracts from Drosophila melanogaster. While several proteins are associated with the translational control element in the RNA, only one of these proteins directly crosslinks to the sequence element. The binding activity is exclusively observed with testis protein extracts but can be demonstrated with testis extracts from other Drosophila species as well, indicating that regulatory proteins involved in translational regulation in the male germ line are conserved. Although binding to the TCE can occur independent of its position relative to the transcription start site of the in vitro transcripts, its function in vivo is not exerted when shifted further downstream within the 5′ UTR of a fusion gene. In addition to being a translational control element the TCE also functions as a transcriptional regulator. Consequently, a DNA‐protein complex is also formed at the TCE. In contrast to the RNA‐protein complexes we find DNA‐protein complexes with protein extracts of several tissues of Drosophila melanogaster. © 1993 Wiley‐Liss, Inc.
Regulated polyadenylation of clam maternal mRNAs in vitro
Authors: Standart Nancy , Dale Martin
Published: May 2005

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA‐specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle‐otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence‐specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu‐cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis‐acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley‐Liss, Inc.
Homeotic gene expression in the locustSchistocerca: An antibody that detects conserved epitopes in ultrabithorax and abdominal‐A proteins
Authors: Kelsh Robert , Weinzierl Robert O. J. , White Robert A. H. , Akam Michael
Published: May 2005

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects,Drosophila(Diptera) andSchistocerca(Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both theUltrabithorax (Ubx)andabdominal‐A (abd‐A)genes inDrosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen ofSchistocerca.We infer that it recognizes theSchistocercahomolog of UBX protein, and probably also of ABD‐A. As the distribution ofSchistocercaABD‐A protein is already known, we can use this reagent to map the expression ofSchistocercaUBX in the thorax and anterior abdomen, where ABD‐A is not expressed. Both the general domain, and many of the details, of UBX exp ression are remarkably conserved compared withDrosophila.Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes inSchistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogene‐sis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. Inthis limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation.

Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups. © 1994 Wiley‐Liss, Inc.
Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects
Authors: Sommer Ralf J. , Tautz Diethard
Published: May 2005

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail‐ related gene fragments from the flour beetle Tri‐bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley‐Liss, Inc.
Heterochronic expression of an adult muscle actin gene during ascidian larval development
Authors: Swalla Billie J. , White Mary E. , Zhou Jing , Jeffery William R.
Published: May 2005

Adultation is a hetercchronic mode of development in which adult tissues and organs differentiate precociously during the larval phase. We have investigated the expression of an adult muscle actin gene during adultation in the ascidian Molgula citrina. Ascidians contain multiple muscle actin genes which are expressed in the larva, the adult, or during both phases of the life cycle. In ascidian species with conventional larval development, the larval mesenchyme cells, which are believed to be progenitors of the adult mesoderm, remain undifferentiated and do not express the muscle actin genes. In M. citrina, the mesen‐chyme cells differentiate precociously during larval development, suggesting a role in adultation. An adult muscle actin gene from M. citrina was obtained by screening a mantle cDNA library with a probe containing the coding region of SpMAl, a Styela plicata adult muscle actin gene. The screen yielded a cDNA clone designated McMAl, which contained virtually the complete coding and 3′ noncoding regions of a muscle actin gene. The deduced McMAl and SpMAl proteins exhibit 97% identity in amino acid sequence and may be encoded by homologous genes. The McMAl gene is expressed in juveniles and adults, but not in larval tail muscle cells, suggesting that it is an adult muscle actin gene. In situ hybridization with a 3′ non‐coding region probe was used to determine whether the McMAl gene is expressed during adultation in M. citrina. McMAl mRNA was first detected exclusively in the mesenchyme cells during the late tailbud stage and continued to accumulate in these cells during their migration into the future body cavity and heart primordium in the hatched larva. The McMAl transcripts persisted in mesenchyme cells after larval metamorphosis. It is concluded that an adult muscle actin gene shows a heterochronic shift of expression into the larval phase during adultation in M. citrina.
Establishment of the dorsoventral axis in nemertean embryos: Evolutionary considerations of spiralian development
Authors: Henry Jonathan Q. , Martindale Mark Q.
Published: May 2005

The Nemertea represent one of a number of invertebrate phyla that display a highly conserved pattern of cell division known as spiral cleavage. The fates of the early blastomeres are known for representatives of some spiralian phyla (i.e., molluscs and annelids) and in these species there appears to be a high degree of conservation in the ultimate fates of particular embryonic cells. The first two cleavage planes bear an invariant relationship to the symmetry properties of the future larval and adult body plan. To investigate whether these properties of spiralian embryo‐genesis are shared (conserved) amongst members of other spiralian phyla, individual blastomeres in two‐ and four‐cell embryos of the nemertean, Nemertopsis bivittata, were microinjected with bi‐otinylated dextran lineage tracers. N. bivittata is a direct‐developing hoplonemertean that forms a nonfeeding larva. When individual blastomeres are injected at the two‐cell stage, two sets of complementary labeling patterns (a total of four different patterns) were observed in the ectoderm of the larvae. When cells were injected at the four‐cell stage, four different patterns were observed that represented subsets of the four patterns observed in the previous experiment. Unlike the case in the annelids and molluscs, in which the first cleavage plane bears a strict 45° angular relationship to the future dorsoventral axis, the first cleavage plane in N. bivittata can bear one of two different relationships relative to the larval/adult dorsoventral axis. In half the cases examined, the first cleavage plane corresponded roughly to the plane of bilateral symmetry, and in the rest, it lay along a frontal plane. A similar result was observed for the embryos of the indirect‐developing heteronemertean, Cerebratutus lacteus. These results indicate that the fates of the four cell quadrants in nemerteans are not directly homologous to those in other spira‐lians, such as the annelids and molluscs. For instance, no single cell quadrant appears to contribute a greater share to the formation of ectoderm, as is the case in the formation of the post‐trochal region by the D‐cell quadrant in annelids and mol‐luscs. Rather, two adjacent cell quadrants contribute nearly equally to the formation of dorsal or ventral ectoderm in the larvae. Possible explanations for the determination of dorsoventrality in nemerte‐ans, as well as implications of these findings regarding the evolution of spiralian development, are discussed. © 1994 Wiley‐Liss, Inc.
IME1 gene encodes a transcription factor which is required to induce meiosis in Saccharomyces cerevisiae
Authors: Mandel Silvia , Robzyk Kenneth , Kassir Yona
Published: May 2005

Previous studies have shown that the IME1 gene is required for sporulation and the expression of meiosis specific genes in Saccharomyces cerevisiae. However, sequence analysis has not revealed the precise functional role of the Ime1 protein. By engineering constructs which express various portions of the Ime1p fused to either the DNA binding or transcriptional activation domains of GAL4, we have conclusively demonstrated that IME1 is a transcription factor, apparently required for sporulation to activate the transcription of meiosis specific genes. The full Ime1p, when fused to the GAL4 DNA binding domain, can both activate GAL1‐IacZ expression, and complement Ime1‐0 (a null allele) for the ability to sporulate, and transcriptionally activate IME2, a meiosis specific gene. As successively larger portions of the encoded Ime1p N‐terminus are deleted from the GAL4(bd)‐IME1 construct, the encoded fusion proteins retain the ability to complement an ime1 null allele, despite a decreasing ability to activate GAL1‐lacZ transcription. However, a fusion construct which retains only the last 45 C‐terminal amino acids of IME1 provides neither transcriptional activation of GAL1‐lacZ nor complementation of ime1‐0. Fusion of a GAL4 activation domain to this portion of IME1, results in a construct with a restored ability to complement an ime1‐0 cllele. This restored ability is dependent upon galactose induction. We conclude, therefore, that IME1 functions in meiosis as a transcriptional activator. © 1994 Wiley‐Liss, Inc.
Isolation of silencer‐containing sequences causing a tissue‐specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster
Authors: Kirkpatrick Robert B. , Parveen Zahida , Martin Presley F.
Published: May 2005

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4‐kb genomic clone containing the 3.2‐kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue‐specific pattern resembling the abnormal expression pattern of the position effect. The 3.2‐kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2‐kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2‐kb fragment indicates that it is a noncoding region. A 17‐bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2‐kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α1‐tubulin promoter‐LacZ fusion construct or an hsp70 promoter‐Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC‐1 line. © 1994 Wiley‐Liss, Inc.