bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization and developmental timing of the Bombyx mori chorion gene clusters in strain C108
Authors: Goldsmith Marian R.
Published: May 2005

We have standardized the map of chorion structural gene clusters in Bombyx mori strain C108 by analyzing quantitative and qualitative chorion electrophoretic markers in recombinant progeny from four independent crosses. In all we assigned 22 markers to three gene clusters, representing about one‐third of the total number of chorion genes: 2 to Ch 1, 9 to Ch 2, and 8 to Ch 3. Three additional markers belong either to Ch 7 or Ch 2. By referring to published chorion protein synthesis patterns, we show that the clusters are restricted in their developmental specificities: Ch 3 appears to be an early locus, carrying all of the mapped early markers (4) and half of the early middles (3/6), while Ch 1 and Ch 2 carry predominantly middle (4/5) and all late, Hc (6) markers, along with some early middle markers (3). We cite evidence to show that Ch 1 and Ch 2 compose the left and right halves of a single gene cluster, which we formally designate as Ch 1–2.
Use of promoter fusions in Drosophila genetics: Characterization of a YP1‐ADH fusion gene
Authors: Aprison Barry S. , Osterbur David L. , Bonner J. Jose
Published: May 2005

In Drosophila melanogaster the yolk protein (YP) genes are normally expressed only in the fat body and follicular epithelium of adult females‐never in males or in larvae. We describe here a first step toward a genetic examination of the developmental controls that restrict the activity of the YP genes to adult female tissues. A YP1 promoter that contains the tissue‐, temporal‐, and sex‐specific controlling elements for expression was fused to the reporter gene, alcohol dehy‐drogenase (Adh). The gene fusion was transformed into an Adh‐deficient genotype. As assayed by a number of criteria, that the fusion gene is expressed in the same physiological manner as the endogenous yolk protein genes. The fusion gene's activity is modulated in trans by a temperature‐sensitive allele of the sex determination gene, tra‐2. The Adh enzyme serves as a selectable marker and therefore these flies are suitable for use in genetic screens for trons‐acting mutations that affect the expression of the yolk protein genes.
Set of proteins shows abnormal posttranslational modification in embryos homozygous for dominant T‐mutations
Authors: Park C.‐H. Thomas , Artzt Karen , Bennett Dorothea
Published: May 2005

T and Tc are dominant mutations in the mouse that affect neuroaxial development when heterozygous and cause embryonic death when homozygous. Embryos were analyzed individually by two‐dimensional gel electrophoresis at 9½ days gestation, 1 day before homozygotes die in utero. A comparison of the protein patterns of mutant homozygotes with those of their littermates revealed a set of proteins (T‐proteins) that showed isoelectric point (pl) polymorphism. All the T‐proteins were more basic in mutant homozygotes. These polymorphisms could be detected, although they were less pronounced, in embryos as young as 7½‐day presomite stages, when it is impossible to distinguish homozygous mutants grossly. Interestingly, the same proteins show a pl shift from basic to acidic in wild‐type embryos during development from 7½ to 9½ days. Thus, it appears that in T and Tc mutants a developmentally specific posttranslational acidic modification of these proteins is disturbed. The likely cause of the abnormality is a defect in some mechanism for phosphorylation, since the T‐proteins of wild‐type embryos were shifted to higher pls by phosphatase treatment. This disturbance appears to be localized to axial structures (neural tube, somites, and surrounding mesenchyme) since only these structures, and not the rest of the mutant homozygous embryos, contain abnormally basic T‐proteins.
Dominant flightless phenotype of the vestigial‐Depilate deficiency of Drosophila melanogaster
Authors: Cripps Richard M. , Sparrow John C.
Published: May 2005

This paper describes the flightless phenotype of the vestigial‐Depilate deficiency of Drosophila melanogaster. Recombination experiments and studies of revertants show that the dominant flightless and depilate phenotypes are inseparable from the deficiency and due to a single cause. Dosage studies on this region reveal that these phenotypes are due to antimorphic effects, probably on the Suppressor‐2 of zeste or Posteriorsexcomb genes which lie close to the distal breakpoint of the deficiency. The deficiency does not uncover a gene haplo‐insufficient for flight. A detailed phenotypic examination failed to reveal any effects of this mutation on the indirect flight muscles. Dr(2R)vgD/+ heterozygotes are unable to initiate flight or raise their wings, even during death by over etherisation. There is a close correlation between the dominant antimorphic flightlessness and patterned thoracic bristle loss which is revealed in interactions with Df(2R)vg62 and the Su(z)2alleles. This is discussed in the light of the bnstle loss mutants of the Achaete‐scute complex. It is proposed that the vestigial‐Depilate deficiency affects the development of thoracic nerves.
Stage‐specific gene expression in early chick embryo
Authors: Zagris Nikolas , Matthopoulos Demetrios
Published: May 2005

Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication inteferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.
Expression of a methotrexate resistant dihydrofolate reductase gene in transgenic mice
Authors: Isola Luis M. , Gordon Jon W.
Published: May 2005

We have previously demonstrated systemic resistance to methotrexate (MTX) in transgenic mice carrying a foreign, mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3) gene. The new gene was introduced as a cDNA cloned into an expression vector driven by the simian virus 40 (SV40) early promoter. Previous physiologic studies suggested that transgenic mice tolerated drug doses invariably lethal to controls on the basis of gastrointestinal (GI) resistance to MTX. In the present study we evaluated foreign gene expression at the RNA level in the three major sites of MTX toxicity: intestine, liver, and bone marrow.

The transgene was transcriptionally active in small bowel, and levels of expression were high in animals tolerating the largest doses of MTX. The gene was also expressed in the liver in some pedigrees, but was not detected in hemopoietic tissues of any of the pedigrees tested. Our studies correlate the site of expression of a drug resistant dhfr gene with an altered physiologic response to MTX, and demonstrate that transgenic mice can be used as a test system for expression of genes considered for use in somatic gene therapy.
Stepwise aggregation, compaction, and differentiation of uncompacted F9 cells
Authors: Littlefield John W.
Published: May 2005

To study the relationship between compaction and differentiation in aggregates of F9 embryonal carcinoma cells, a subline was developed which grows mostly uncompacted in monolayer culture in medium containing a low concentration of calcium (about 0.05 mM). When these cells were trypsinized and cultured in suspension in the same medium, they formed loose, open aggregates, which failed to differentiate into embryoid bodies after exposure to 10 nM retinoic acid, confirming the requirement of compaction for differentiation. If, after culture for 3 days, the uncompacted F9 aggregates were exposed to additional calcium (4 mM), all compacted within an hour. The number of days necessary for aggregates to acquire this ability to compact rapidly was reduced if the monolayer of cells from which the aggregates were derived had been exposed to additional calcium to cause compaction for several days prior to trypsinization and aggregation. Next, treatment of the compacted F9 aggregates with 10 nM retinoic acid was followed by differentiation into embryoid bodies. The number of days required for this was also reduced if the aggregates were formed from previously compacted cells, presumably because compaction of the aggregates occured sooner.

The acceleration in compaction and differentiation in aggregates formed from previously compacted cells suggests that some of the proteins important for compaction, which are synthesized in a monolayer of compacted cells, persist through trypsinization and are carried over from monolayer to aggregates. Alternatively, an inhibitor of compaction is decreased in the compacted monolayer. Thus, the process of compaction in its entirety, including its relationship to subsequent differentiation, cannot be studied in aggregates formed from F9 cells grown as usual in the compacted state in monolayer culture. This work provides an alternative system in which aggregation, compaction, and differentiation of F9 cells can be made to occur in stepwise fashion and can be examined separately.
Characterization of bz1 mutants isolated from mutator stocks with high and low numbers of Mu1 elements
Authors: Hardeman Kristine J. , Chandler Vicki L.
Published: May 2005

The high frequency of mutations in Mutator stocks of maize is the result of transposition of Mu elements. Nine different Mu elements that share the 220 bp Mu terminal inverted repeats have been described. Mul elements have been found inserted into most of the molecularly characterized mutant alleles isolated from Mutator stocks, and most Mutator stocks contain a high number of Mul elements (10‐60). However, it is clear that additional Mu elements, which share the Mul termini but have unrelated internal sequences, can also transpose in Mutator stocks. We were interested in comparing the mutation frequency and type of elements that inserted into a particular locus when Mutator stocks with differing numbers of Mul elements were utilized. Furthermore, previous studies with Mu‐induced mutations have demonstrated that the element that inserted most frequently was Mul. Therefore, to try to obtain Mu elements different from Mul we utilized a stock that had a low number (3‐6) of Mul elements as well as a Mutator stock with a more typical number of Mul elements (20‐60).

Utilizing both stocks, we isolated numerous mutants at one gene, Bronze1 (Bz1), and compared the type of elements inserted. In this paper we report that both the high and low Mu1 stocks produced bz1 mutants at frequencies characteristic of Mutator stocks, 6.6 and 4.3 ± 10−5, respectively. We describe the isolation of 20 bz1 mutations, and the initial molecular characterization of eight unstable mutations: two from the high Mu1 stock and six from the low Mu1 stock. The six alleles isolated from the low Mu1 stock appear to contain deleted Mu1 elements, and the two alleles isolated from the high Mu1 stock contain elements very similar to Mu1. When the mutants from the low Mu1 stocks were examined, it was found that the Mu1 ‐related elements increased from 3‐6 copies to 9‐20 copies in one generation. The high number of Mu1 ‐related elements was maintained in subsequent out‐crosses. This spontaneous activation and amplification of Mu ‐related elements occurred in at least 1% of the low Mu1 plants.
Quartet: A Drosophila developmental mutation affecting chromosome separation in mitosis
Authors: Zahner Joseph E. , Cheney Clarissa M.
Published: May 2005

The Drosophila mutation, quartet, affects development at points in the life cycle that require intense mitotic activity. Examination of embryos affected by the maternal effect of quartet has revealed defects that can be attributed to incomplete chromosome separation at mitosis. These defects include uneven spacing of nuclei, strands of DNA creating bridges between nuclei, and abnormal amounts of DNA per nucleus. Nuclei in quartet‐affected embryos also have a greater‐than‐normal number of centrosomes. Immunofluorescent examination of the spindles in quartet‐affected embryos has revealed tripolar spindles and adjacent spindles that share a common spindle pole. Finally, chromosome separation distance was measured in anaphase and telophase spindles in quartet‐affected embryos and found to be blocked in anaphase. Examination of mitotic figures in quartet larvae revealed a reduced mitotic index and an elevated frequency of abnormal mitotic figures. quartet could encode a function necessary for the disengagement of chromosomes in mitosis, for kinetochore function or for function of a spindle motor. Mutations in quartet prevent the post‐translational modification of three abundant proteins. These proteins may be involved in chromosome separation in mitosis.
Genetic alteration of normal aging processes is responsible for extended longevity in Drosophila
Authors: Arking Robert , Wells Robert A.
Published: May 2005

The first step in a genetic analysis of aging is to identify and characterize the genetic mutants and their controls that will be used. Such mutants or strains are initially identified by their effect on the life span. Yet many genetic interventions are known to have some effect on the life span without necessarily affecting the aging process. It is therefore necessary to prove that one is actually dealing with an aging mutant before one draws strong inferences from the data. Casarett's rules provide an operational test for doing so, relying as they do on the comparison of aging biomarkers in the experimental and reference strains. We show that our previously described genetically based long‐lived NDC‐L strain and its normal‐lived NDC‐R control strain differ only in the chronological age of expression of two behavioral and three physiological functional age biomarkers. They do not differ in the sequence or the physiological age of expression of these biomarkers. These two strains comply with the Casarett rules and thereby comprise a valid tool with which to conduct a comparative genetic analysis of aging. The implications of the available data are discussed, including the possibility that aging in these strains of Drosophila melanogaster may be the result of a multiphasic developmental process.
Chimeric genes and transgenic plants are used to study the regulation of genes involved in symbiotic plant‐microbe interactions (nodulin genes)
Authors: de Bruijn Frans J. , Szabados Laszló , Schell Jeff
Published: May 2005

Nodulin genes are plant genes specifically activated during the formation of nitrogen‐fixing nodules on leguminous plants. These genes are interesting to study since they are not only induced in a specific developmental fashion by signals coming directly or indirectly from the rhizobial symbiont, but are also expressed in a tissue‐specific manner. By examining the expression of chimeric nodulin‐reporter genes in transgenic legume plants it has been shown that nodule specific expression is mediated by DNA sequences present in the 5′upstream region of several nodulin genes. Here we summarize the available data on these cis‐acting elements and the trans‐acting factors interacting with them. We also review experiments designed to identify rhizobial “signals” which may play a role in nodule specific gene expression.
Regulation of nuclear genes encoding chloroplast proteins in transgenic plants
Authors: Fluhr Robert
Published: May 2005

Transgenic plants have been particularly useful in studying nuclear genes encoding for photosynthetic functions. The expression of these genes and their chimeric constructs in transgenic plants faithfully mimics their natural counterparts. The use of sensitive chimeric reporter genes has enabled localizing the activity of genes encoding photosynthetic proteins to individual cells. Cab and rbcS transgenes have been shown to retain sensitivity to light quality, which is modulated by phytochrome. Conditional light activation under the influence of a circadian rhythm has been shown for Cab transgenes. Transgenic plants containing truncated promoters have helped delineate cis‐regulatory positive and negative elements involved in light‐mediated transcriptional induction and tissue specificity.
Gametic imprinting and the manifestation of the fused gene in the house mouse
Authors: Ruvinsky Anatoly O. , Agulnik Alexander I.
Published: May 2005

The phenomenon of gametic imprinting in mammals has raised developmentally relevant questions concerning the manifestation and inheritance of genes with variable penetrance. The dominant fused (Fu) gene located on chromosome 17 is one of the few good cases demonstrating the phenomenon in mice. The Fu mutation has a maternal effect.

We have previously shown that the † 12 haplotype significantly lowers the penetrance of Fuin ♀ ♀ Fu/†12 offspring. Results of recipiocal matings of the heterozygotes for Fu indicated that the Fu of maternal origin has a lowered level of penetrance. The dominant suppressors locotad outside chromosome 17, in contrast to †12 residing in it, had stronger effects on the manifestation of Fu, decreasing its penetrance to 8–17%. Experimental evidence is presented that the pathway via which Fu passes to the zygote nucleus during gametogenesis through successive generations has a marked effect on its penetrance. Based on this evidence, patterns of genetic imprinting are described. A survey of genetic imprinting allowed us to distinguish two developmental phases, gametic and zygotic. The hypothesis for the gametic phase of the development of multicellular organisms suggests that it proceeds from initialization, a process thought to ensure the freeing of chromosomes from redundant epigenetic information and their preparation for the consecutive developmental cycle.
Recent advances in the molecular genetics of Dictyostelium
Authors: Knecht D. A. , Kessin R. H.
Published: May 2005

DNA mediated transformation is a critical technique for uniting genetic, molecular genetic and reverse genetic approaches to a wide range of problems in cell and molecular biology. In Dictyostelium, there is now the capability not only to manipulate DNA sequences in vitro and put them back into the cell, but also to alter the sequences of endogenous chromosomal genes through high frequency homologous recombination. This means that the range of gene manipulation techniques that have made yeast such a useful system should now be applicable in Dictyostelium. For studying problems such as cellular motility, morphogenesis, and gene regulation, few organisms have the combination of features offered by Dictyostelium.
Establishment of conditions for the transformation of nonaxenic Dictyostelium strains
Authors: Lloyd Malgorzata M. , Ceccarelli Adriano , Williams Jeffrey G.
Published: May 2005

We have studied the transient expression, in Dictyostelium cells growing on a bacterial food source, of a construct containing the coding region of the firefly luciferase gene inserted downstream of a Dictyosteliumactin promoter. The fusion gene is not detectably expressed when DNA is introduced by calcium phosphate precipitation or by electroporation, but it is expressed when introduced using cationic liposomes (lipofectin). Using this latter procedure, we are able to transform cells with a G418 resistance vector and select stable, drug‐resistant transformants at a relatively low, but workable, efficiency. This technique will allow molecular genetics to be applied to the many important nonaxenic Dictyostelium strains.
Regulatory gene interactions controlling discoidin lectin expression in Dictyostelium discoideum
Authors: Alexander Stephen , Leone Sandra , Ostermeyer Elizabeth , Sydow Lisa M.
Published: May 2005

Genetic studies have revealed a network of three unlinked regulatory genes that control the developmental expression of the family of endogenous lectins in Dictyostelium discoideum. Mutations in the disA and disB loci have a null phenotype and do not express the discoidin I or II lectins. The third mutation, drsA, is a second‐site suppressor of the disB mutation, which restores expression of all lectin species. Cells carrying this mutation express the discoidin lectins during growth, which is in contrast to wild‐type cells in which lectin synthesis is developmentally regulated. In addition to this basic level of genetic control, the conditions of growth dramatically influence the patterns of discoidin expression. Growth‐phase wild‐type cells do not express lectin if the cells are grown on plates in association with bacteria. However, wild‐type cells growing in bacterial suspensions express high levels of lectin during growth. Synthesis of the discoidin lectins in growing cells is sensitive to the levels of extracellular cyclic adenosine monophosphate (cAMP) and folic acid. These results suggest that the drsA mutation renders cells insensitive to cAMP and/or folate and thus allows expression of lectin during growth.
Homology cloning of protein kinase and phosphoprotein phosphatase sequences of Dictyostelium discoideum
Authors: Haribabu Bodduluri , Dottin Robert P.
Published: May 2005

Reversible protein phosphory‐lation appears to be important of several stages in the signal transduction pathways in Dictyostelium discoideum. To elucidate its role, we have isolated sequences encoding putative protein kinases and phosphoprotein phosphatases by homology clon‐ ing using polymerase chain reactions (PCRs). Oli‐gonucleotide primers were synthesized for use as forward and reverse primers with their nucleotide sequences deduced from the amino acid sequences of conserved domains of several protein kinases and phosphoprotein phosphatases. The fragments amplified by PCR were cloned, sequenced, and shown to encode parts of five different protein kinases and two phosphoprotein phos‐phatases. Several features such as the deduced amino acid sequence homology, location of invariant amino acids, GC content, and the codon usage confirmed that one set of clones encode parts of different protein kinases of Dictyostelium. Two clones derived from phosphoprotein phosphatase primers encode fragments of type 1 and type 2A phosphoprotein phosphatases. Amplified fragments were used to screen a Xgtll bank, and several cDNA clones for protein kinases were isolated. Some of these show differential expression during development or in response to exogenous cAMP.
Gene expression and chromatin structure in the cellular slime mold, Dictyostelium discoideum
Authors: Blumberg Daphne D. , Agarwal Ameeta K. , Sloger Marcia S. , Yoder Bradley K.
Published: May 2005

Micrococcal nuclease digestion of chromatin from growing cells reveals a structural organization which differs for genes transcribed at diverse rates. The late cAMP dependent prespore genes which are not transcribed in growing cells are found in growing cells in a regular nucleosomal repeat with an average spacing of 168 nucleotides. By contrast genes expressed at a low level in growing cells show an irregular pattern of bands with an average distance between bands of 80 nucleotides. The sizes of the bands generated from the transcribed genes are consistent with the concept that transcription results in the loss of the linker region histone H1 with concomitant sliding of nucleosomes to generate close packed (“slipped”) di, tri, and tetra nucleosomes lacking the linker region. Further analysis of dinucleosomes released by micrococcal nuclease digestion reveals that transcriptionally active genes are found associated with dinucleosome species which may be lacking histone H1. The length of DNA protected by these dinucleosomes is heterogeneous, ranging from 250 to 300 nucleotides.

Methodology is described which has been adapted to allow two dimensional hybridization mapping of nucleoprotein complexes on single copy Dictyostelium genes.
Analysis of the multiple transcripts of the Dd ras gene during Dictyostezium discoideum development
Authors: Reymond Christophe D. , Thompson Nancy A.
Published: May 2005

Transcripts from the Dd ras gene can only be detected once starved cells have begun to aggregate (Reymond et al., Cell 39: 141–148, 1984). We show in this report that the three transcripts which originate from Dd ras during nor‐ mal development differ in their 5′ ends. In suspen‐ sion of starved single cells, one major Dd ras RNA accumulates upon addition of CAMP. It seems that the CAMPregulation of Dd ras expression happens both at the transcriptional and post‐transcriptional level. An RNA secondary structure present in the 5′ untranslated region of the gene is proposed to be important in this post‐transcriptional regulation.
The relative importance of transcriptional and post transcriptional regulation of Drosophila chorion gene expression during oogenesis
Authors: Romano Charles P. , Martinez‐Cruzado Juan Carlos , Kafatos Fotis C.
Published: May 2005

To determine the relative roles of transcriptional and post‐transcriptional events in establishing the temporal pattern of chorion gene expression in Drosophila, we have examined chorion gene transcription, RNA accumulation, and protein synthesis in follicles of selected pre‐early‐ and late‐choriogenic stages. Chorion gene transcription was assayed in follicle cell nuclei by nuclear run‐on reactions. For the s 15, s 16, s 18, s36, and s38 chorion genes, the periods of intense transcription are as predicted from the dynamics of RNA accumulation and protein synthesis, indicating that these genes are primarily regulated at the transcriptional level. In contrast, gene s19 appears subject to post‐transcriptional control at stage 14, when transcription rates are substantially higher than predicted from the observed RNA levels.

Transcription of regions between the clustered and tandemly oriented chorion genes was also examined. In contrast to many RNA polymerase II transcribed genes, for the s18 and s36 chorion genes run‐on transcription appears to terminate within about 100 base pairs downstream of the polyadenylation sites, corroborating previous reports based on electron microscopy of s36 [Osheim et al., EMBO J 5:3591–3596, 1986].
Developmental regulation of the sheep β‐lactoglobulin gene in the mammary gland of transgenic mice
Authors: Harris S. , McClenaghan M. , Simons J. P. , Ali S. , Clark A. J.
Published: May 2005

β‐Lactoglobulin (BLG) is the most abundant whey protein in sheep milk but it is not present in mouse milk. We have previously shown that transgenic mice carrying the BLG gene express it specifically in the mammary gland and secrete BLG into milk at high concentrations. Here we demonstrate that BLG transcription is correctly initiated in mice and that BLG synthesis is restricted to the secretory epithelial cells of the mammary gland. We have also determined the temporal pattern of milk protein gene expression and find that the BLG transgene is regulated coordinately with mouse β‐casein and that the patterns of regulation of BLG in mouse and sheep share some similarities.
Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice
Authors: Iba Koh , Takamiya Ken‐Ichiro , Toh Yoshihiro , Satoh Hikaru , Nishimura Mitsuo
Published: May 2005

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non‐allelic temperature‐sensitive virescent mutants of rice. Temperature‐shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast‐encoded genes but do not affect the subsequent morphological development of the leaf or the photo‐regulation of the expression of nuclear genes encoding chloroplast proteins.
Embryonic mutants of Arabidopsis thaliana
Authors: Meinke David W.
Published: May 2005

Genetic analysis of plant em‐bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T‐DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le‐thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley‐Liss, Inc.
In vitro transcription in isolated nucleoli ofTetrahymena pyriformis
Authors: Matsuura Tadashi , Higashinakagawa Toru
Published: May 2005

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre‐rRNA) and nascent pre‐rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5′ region of their 35S pre‐rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of tran‐scriptional control during development and differentiation. © 1992 Wiley‐Liss, Inc.
Spatial regulation in the expression of structural and regulatory storage‐protein genes in Zea mays endosperm
Authors: Dolfini Silvana Faccio , Landoni Michela , Tonelli Chiara , Bernard Loris , Viotti Angelo
Published: May 2005

Endosperm development in maize seed involves the multiplication, enlargement, and differentiation of cells with consequent accumulation of storage products. The storage protein genes, encoding zeins, and glutelins (multigene families) are expressed and developmentally regulated by different loci. Wild‐type lines and genotypes carrying mutations at loci affecting zein synthesis (o2, o7, fl2, and prol) were characterized at the molecular level and investigated by Northern analysis in order to define the expression of structural and regulatory genes. In situ hybridization in both wild‐type and mutant lines was performed to visualize the spatial distribution of transcripts representing each gene family, during endosperm development. The zein and glutelin mRNAs are expressed in all endosperm cells, except for the aleurone layer. However, each mRNA type accumulates at a different level in the various endosperm regions, thus allowing to recognize specific territories of expression for each storage protein mRNA within the tissue. The spatial expression patterns appear early for each gene type and are maintained during the course of endosperm development. Also, the quantitative distribution of the same transcripts in endosperm of mutant lines is specific for each mutant and different from that of the wild‐type. Furthermore, the amount of the O2 transcript, present in the nucleus and cytoplasm of wild‐type cells, varies substantially in the different o2 mutations considered, in one mutant almost exclusively confined within the nucleus. These data suggest a specific control of the spatial expression of storage protein genes and a heterogeneous molecular composition of protein bodies throughout the endosperm tissue. © 1992 Wiley‐Liss, Inc.
Developmental regulation of excision timing of Mutator transposons of maize: Comparison of standard lines and an early excision bzl::Mu1 line
Authors: Walbot Virginia
Published: May 2005

Three characteristics of standard Mutator lines reflect developmental regulation: new mutants usually involve single gametes, somatic excision is restricted to terminal cell divisions during tissue development, and germinal excision is rare. By selection for earlier (larger) somatic sectors in the aleurone, a Mutator line was identified that exhibits a dramatic elevation in somatic excision frequency during the first three nuclear divisions of the endosperm and more than a 10‐fold increase in germinal reversion from the bzl::Mul reporter gene. The programming of early sectoring is dominant in crosses with Mutator lines containing diverse reporter alleles. Germinal reversion is biased 5‐ to 10‐fold for events through the pollen compared to the ear. The timing of germinal excision in the tassel is late because somatic excision sectors in the anthers are small; however, 98% of the germinal revertants are concordant. These observations indicate that in the early sectoring line Mu excision usually occurs before the mitotic divisions that separate gametic nuclei and may be restricted to the early stages of microsporogenesis. © 1992 Wiley‐Liss, Inc.
Ferritin gene expression is developmentally regulated and induced by heat shock in sea urchin embryos
Authors: Infante A. A. , Infante D. , Rimland J.
Published: May 2005

A 20‐kD protein identified as a subunit of the iron‐binding protein ferritin is present in S. purpuratus and L. pictus sea urchin embryos. The synthesis of the protein is stimulated by an elevation in temperature or by an increase in iron supply. The developmental expression of this protein and its regulation during normal development and upon heat shock was investigated. In L. pictus, ferritin is present in the unfertilized egg and, as determined by Western blot analysis, its concentration remains approximately constant after fertilization up to the gastrulc‐pluteus stage; there is a small transient decrease in the level of the protein in the early blastula at a time coinciding with the first clear indication of its de novo synthesis. Northern blots reveal no cytoplasmic ferritin transcripts in the unfertilized egg, but there occurs a dramatic increase in the RNA level from the late morulaearly blastula stage (12–14 hr) to the mesenchyme blastula‐early gastrula (25–30 hr) stage. This developmentally regulated increase in the constitutive concentration of ferritin RNA is correlatable with the normal onset of synthesis of the protein. The overall degree and nature of induction of ferritin by heat is dependent on the developmental stage: at 10–16 hr postfertilization heat shock elicits an increase in both the concentration of RNA and the synthesis of the protein; in hatched blastula (18 hr) and in later embryos heat shock increases ferritin synthesis, without a corresponding increase in the mRNA level. It appears that different mechanisms operate in the developing sea urchin embryo to regulate the expression of ferritin during normal development and on exposure to heat stress, one dependent on the concentration of ferritin transcripts and another operating at the level of translational control. © 1993Wiley‐Liss, Inc.
Heat shock induces changes in the expression and binding of ubiquitin in senescentDrosophila melanogaster
Authors: Niedzwiecki Aleksandra , Fleming James E.
Published: May 2005

We examined the effect of aging on the expression of ubiquitin RNA and the binding of the ubiquitin polypeptide to proteins following heat shock inDrosophila melanogaster.Heat‐shocked adult flies transcribe two major RNA species‐one of 4.4 kb and one of about 6 kb that hybridize to the polyubiquitin‐encoding probe. Several less abundant RNAs were also observed but the 4.4‐kb band was present as the major RNA species in both stressed and nonstressed flies of both ages. The 6‐kb fragment was more abundant in heat shocked aged flies than in younger flies. The quantitative expression of the polyubiquitin gene increased in proportion to the duration of the heat stress. Moreover, the induction of the polyubiquitin RNA was markedly elevated during aging following heat shock. Hybridization of Northern blots with the monoubiquitin gene probe revealed a band of 0.9 kb that was not significantly affected by heat stress.

We also investigated the relationship between the changes in polyubiquitin gene expression and the formation of ubiquitin‐protein complexes in aging heat‐shocked flies. Heat shock to old flies results in a significant increase in the level of proteins immunoprecipitated by anti‐ubiquitin antibodies. In the case of proteins synthesized 2 h before heat shock, most of the ubiquitinated proteins were of high molecular weight. For those proteins synthesized during a 30‐min heat shock and the 2 h following heat shock, two major immunoprecipitated bands were observed: an 80‐kD and a 70‐kD polypeptide. The ubiquitination of a 60 kD protein was also observed in nonstressed flies, but its for mation was drastically reduced following heat shock. For proteins synthesized during and after heat shock from both age groups, the major ubiquitinated polypeptide is 70 kD. In all age groups, more ubiquitin complexes were formed with proteins synthesized before heat shock, than with proteins synthesized either during or after heat shock. This suggests that cellular proteins synthesized at physiological temperatures are more sensitive to heat induced damage than those synthesized during stress. These data support the hypothesis that in aging flies, heat shock induces an unusually high concentration of abnormal proteins which are targeted for degradation by the ubiquitin‐dependent proteolytic system. © 1993Wiley‐Liss, Inc.
Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development
Authors: Mosser Dick D. , Duchaine Jean , Bourget Lucie , Martin Luis H.
Published: May 2005

Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4−CD8−, adult thymocytes, which are primarily CD4+CD8+, and mature spleen T cells, which are CD4+CD8− or CD4−CD8+. After either a 41°C or 42°C heat shock, the synthesis of the maior heat‐inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4+CD8+ cells were more sensitive to hyperthermia than either the double negative CD4−CD8− or single positive CD4+CD8− or CD4−CD8+ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome‐sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T‐cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley‐Liss, Inc.
Expression of alternative forms of differentiation inhibiting activity (DIA/LIF) during murine embryogenesis and in neonatal and adult tissues
Authors: Robertson Morag , Chambers Ian , Rathjen Peter , Nichols Jennifer , Smith Austin
Published: May 2005

Differentiation inhibiting activity/leukaemia inhibitory factor (DIA/LIF) is a pleiotropic cytokine which has been implicated in a variety of developmental and physiological processes in mammals due to its broad range of biological activities in vitro. A role in very early development is suggested by the requirement for DIA/ LIF to support the self‐renewal of cultured embryonic stem (ES) cells. Other data point to potential roles in the establishment and maintenance of primordial germ cells, in osteogenesis and in haematopoiesis, and possibly in neuronal specification. DIA/LIF may also act as a mediator of the hepatic acute phase response. In the present study the expression of DIA/LIF transcripts during murine development and in adult mice has been determined using a highly sensitive ribonuclease protection analysis. In contrast to previous reports, it is apparentthat DIA/LIF transcripts are present at low levels in many adult mouse tissues. Higher levels of expression are observed in skin, lung, intestine, and uterus. Elevated amounts of mRNA are also found in certain foetal tissues during late gestation and neonatally. In earlier embryogenesis, however, DIA/LIF mRNA is produced primarily in extraembryonic tissues. The alternative transcripts which produce either soluble or matrix‐associated DIA/LIF exhibit overlapping but non‐identical patterns of expression, consistent with the proposition that the two isoforms may have distinct biological functions. These findings are suggestive of widespread roles for DIA/LIF in vivo and are discussed in the light of available data on the phenotype of homozygous DIA/LIF‐deficient mice. © 1993Wiley‐Liss, Inc.
Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos
Authors: Tadano Takafumi , Otani Hiroki , Taira Masanori , Dawid Igor B.
Published: May 2005

Mesoderm development in Xenopus laevis depends on inductive cell interactions mediated by diffusible molecules. The mesoderm inducer activin is capable of redirecting the development of animal explants both morphologically and biochemically. We have studied the induction of four regulatory genes, Mix. 1, goosecoid (gsc), Xlim‐1 and Xbra in such explants by activin, and the influence of other factors on this induction. Activin induction of gsc is strongly enhanced by dorsalization of the embryo by LiCl, while expression of the other genes is only slightly enhanced. The protein synthesis inhibitor cycloheximide (CHX) inhibits the activin‐dependent induction of Xbra partially, while induction of Mix. 1 and Xlim‐ 1 is essentially unaffected. In contrast, gsc shows strong superinduction in the presence of activin and CHX, and can be induced in animal explants by CHX alone. Induction and superinduction by CHX have previously been observed for immediate early genes in a variety of systems, notably for the activation of c‐fos expression by serum stimulation, but have not been reported in early amphibian embryos. © 1993Wiley‐Liss, Inc.
Attenuation of white gene expression in transgenic Drosophila melanogaster: Possible role of a catalytic antisense RNA
Authors: Heinrich Jörg‐Christian , Tabler Martin , Louis Christos
Published: May 2005

We have transformed Drosophila melanogaster with a DNA construct encoding a catalytic antisense RNA directed against the primary white gene RNA transcript. Total RNA isolated from transformed flies was shown to catalyze the specific cleavage of in vitro transcribed substrate RNA, indicating the expression of a functional ribozyme. Moreover, transgenic lines carrying homozygous copies of this construct depict a further clear‐cut reduction in eye pigmentation when present in a genetic background that has a priori reduced levels of white gene expression. © 1993Wiley‐Liss, Inc.
Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP
Authors: Sucic Joseph F. , Selmin Ornella , Rutherford Charles L.
Published: May 2005

A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp‐2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp‐2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp‐2 mRNA. The induction of gp‐2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 μM could induce gp‐2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp‐2 occurs. Induction of gp‐2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp‐2 gene for cis‐acting elements that are involved in the cAMP regulation of gp‐2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from ‐258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis‐acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5′ end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. © 1993Wiley‐Liss, Inc.
Tissue‐specific regulation by ecdysone: Distinct patterns of Eip28/29 expression are controlled by different ecdysone response elements
Authors: Andres Andrew J. , Cherbas Peter
Published: May 2005

The Eip28/29 gene of Drosophila is an example of a tissue‐ and stage‐specific ecdysone‐responsive gene. Its diverse patterns of expression during the third larval instar and a synopsis of those patterns in terms of expression groups have been reported previously. Here we have studied the expression (in transgenic flies) of reporter genes controlled by Eip28/29‐derived flanking DNA. During the middle and late third instar, most tissues exhibit normal expression patterns when controlled by one of two classes of regulatory sequences. Class A sequences include only 657 Np of 5′ flanking DNA from Eip28/29. Class B sequences include an extended 3′ flanking region and a minimal (≤93 Np) 5′ flanking region. The class B sequences include all those elements known to be important for ecdvsone induction in cultured cells. They are sufficient to direct the normal premetamorphic induction of Eip28/29 in the lymph glands, hemocytes, proventriculus, and Malpighian tubules. This is consistent with our suggestion that Kc cells are derived from embryonic hematopoietic cells. It is remarkable that the epidermis requires only class A sequences. These are sufficient to up‐regulate expression at medinstar and to down‐regulate expression at metamorphosis. It follows that the epidermis uses EcREs distinct from those that function in Kc cells. It is possible that the Upstream EcRE, which is nearly silent in Kc cells, is active in the epidermis. © 1994 Wiley‐Liss, Inc.