bluuebunny/crossref_metadata_2025 huggingface dataset:
Anemia in a line of transgenic mice carrying a mutant dihydrofolate reductase gene
Authors: Isola Luis M. , Gordon Jon W.
Published: May 2005

Several lines of transgenic mice were produced by pronuclear injection of a full‐length cDNA encoding a mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3). The mutation causes altered enzyme kinetics for folate reduction as well as low affinity for methotrexate (MTX). One line of mice carrying the plasmid displays a moderate‐to‐severe anemia that is evident in fetuses and newborn mice and that moderates with age. RNA studies revealed high levels of transcription of the mutant gene in the fetal and adult liver, and low or absent expression in adult bone marrow. Transcription of the mutant gene was not found in the fetal liver of other pedigrees examined. The data thus suggest that expression of this mutant gene in the main hematopoietic organ of the fetus adversely affects erythropoiesis by altering the cellular environment for erythroid differentiation, and that translocation of the site of hematopoiesis to bone marrow, where the foreign gene is not expressed, leads to normalization of red cell production.
Effect of low‐temperature rearing on heat shock protein synthesis and heat sensitivity in Drosophila melanogaster
Authors: Singh A. K. , Lakhotia S. C.
Published: May 2005

The patterns of synthesis of heat shock proteins (hsp) and heat sensitivity to elevated temperatures in larvae of Drosophila melanogaster reared since hatching at 20°C (warmreared) or at 10°C (cold‐reared) were compared. The pattern of hsp synthesis in salivary glands from the cold‐ and warm‐reared late‐third‐instar larvae exposed for l hr to 33°C or to 37°C was generally similar except for remarkable differences in the 23 kd hsp and a heat‐inducible 14 kd polypeptide. The hsp 23 was abundantly synthesised in control as well as heat‐shocked warm‐reared larval salivary glands, its synthesis in heat‐shocked glands being dependent on new transcription. The synthesis of hsp 23 was much less in control glands of cold‐reared larvae and was not further inducible by heat shock. The 14 kd polypeptide synthesis was greater in control as well as heat‐shocked salivary glands of cold‐reared larvae, whereas, in the warm‐reared ones, its activity was much less. The cold‐reared larvae showed greater sensitivity to elevated temperature; fewer adults eclosed when the cold‐reared late‐third‐instar larvae were exposed to 40°C for l hr and also a pretreatment at 37°C for l hr was less effective in stopping the killing effect of a subsequent 40°C heat shock in cold‐reared than in warmA‐reared larvae. The greater thermosensitivity of the cold‐reared larvae may be correlated with the altered patterns of heat shock gene transcription and translation in cold‐reared larvae.
Structure and expression of the cAMP cell‐surface receptor
Authors: Saxe Charles L. , Klein Peter , Sun Tzeli J. , Kimmel Alan R. , Devreotes Peter N.
Published: May 2005

Using antibodies specific for the 3′, 5′‐cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened γgtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: (1) β‐galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, (2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, (3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, (4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and (5) RNA transcribed in vitro from cDNAs containing the entire protein‐coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA.

The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell‐surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G‐proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.
Characterization of an unusual cAMP receptor and its related polypeptides in Dictyostelium discoideum
Authors: Tsang Adrian , Grant Caroline , Kay Carolyn , Bain Gerard , Greenwood Michael , Noce Toshiaki , Tasaka Masao
Published: May 2005

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell‐surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti‐CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co‐purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1‐related polypeptides can be detected on the cell surface as well as in the nucleus.
Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol
Authors: Kimmel Alan R. , Eisen Michael
Published: May 2005

In Dictyostelium, extracellular cAMP interacts specifically with cell‐surface receptors to promote the accumulation of a variety of intracellular second messengers, such as, 3′‐5′ cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell‐surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP‐dependent gene expression can occur in the absence of receptor‐mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor‐mediated activation of a membrane‐bound phosphodiesterase. IP3 and, thus, by inference, diacylglycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell‐surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.
A Ca2+‐dependent signal transduction system participates in coupling expression of some cAMP‐dependent prespore genes to the cell surface receptor
Authors: Blumberg Daphne D. , Comer Joann F. , Higinbotham Kathleen G.
Published: May 2005

Elevated levels of cAMP are essential for the expression of many postaggregation prespore and prestalk mRNA species and for the suppression of some growth phase mRNAs. Here we review evidence that this regulation is mediated by cAMP interacting at the cell surface receptor. These effects of cAMP on gene expression can occur under conditions where the receptor‐associated adenylate cyclase is inactivated and in concentrations that are consistent with receptor binding. A number of differences are noted in the mechanism by which cAMP regulates prespore and prestalk genes. Finally, evidence is reviewed for the role of a Ca2+‐dependent signal transduction system in coupling the expression of some of the prespore mRNAs to the cAMP receptor. This signal transduction system does not appear to be involved in the expression of the cAMP‐dependent prestalk gene.
Analysis of cis and trans elements involved in cAMP‐inducible gene expression in Dictyostelium discoideum
Authors: Hjorth Annegrethe , Datta Sumana , Khanna Navin C. , Firtel Richard A.
Published: May 2005

Expression of the Dictyostelium discoideum pst‐cath (CP2) gene is transcriptionally regulated during multicellular development, and the gene is inducible in competent single cells following administration of exogenous cAMP. The 5′ flanking region of pst‐cath (CP2) that extends from −313 to the Cap site (+−1) has previously been shown to contain sufficient cis,‐acting regulatory elements for proper developmental and cAMP‐inducible expression of a foreign gene [Datta and Firtel, 1987, Mol Cell Biol 7:149–159]. The −283 to −201 region includes two exceptional “G‐boxes” centered at −233 and −217 respectively, and this ∼︁ 80 bp region is essential for basal as well as regulated expression of the pst‐cath (CP2) gene. Here we summarize results obtained from a detailed analysis of a series of linker‐scanner mutants and mutants that carry small internal deletions within the essential 80‐bp region. Insertion of a synthetic oligonucleotide that includes the downstream G‐box is demonstrated to rescue a low level of cAMP‐inducible expression following insertion into cassette mutants. The effect of introducing a change in the relative spacing between regulatory elements has also been investigated.

We have analyzed nuclear extracts for the presence of DNA‐binding proteins that interact specifically with the pst‐cath (CP2) regulatory region and identified two such putative trans‐acting factors: (1) the AT‐factor that is observed within a few hours following the onset of starvation and that binds tightly to stretches of alternating adenine‐thymine residues (poly(dA‐dT)); and (2) the AG‐factor that is present in nuclear extracts of aggregated cells. Competition studies have demonstrated significant differences in the affinity that characterizes the binding of the two factors to G‐box‐containing sequences. The binding specificities of these DNA‐binding proteins have been analyzed using gel mobility‐shift and DNaseI footprinting assays.
Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G‐rich sequence element lying between them with the characteristics of a basal promoter element
Authors: Driscoll Donna M. , Pears Catherine J. , Williams Jeffrey G.
Published: May 2005

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G‐rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP‐inducibility. A synthetic oligonucleotide, containing two copies of the G‐rich element from the CP1 gene, will reconstitute cAMP‐inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP‐inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.
Nuclear plasmids in the Dictyostelium slime molds
Authors: Hughes Joanne E. , Ashktorab Hassan , Welker Dennis L.
Published: May 2005

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2‐μm circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3–27 kb and in copy number from 50‐300 molecules per cell. Plasmids have been identified in approximately one‐fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10−4 per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.
Actin‐binding proteins are conserved from slime molds to man
Authors: Schleicher Michael , André Elisabeth , Hartmann Herbert , Noegel Angelika A.
Published: May 2005

DNA clones encoding the actin‐binding proteins α‐actinin and severin from Dictyostelium discoideum were isolated and sequenced. Comparisons of the deduced amino acid sequences with proteins from other species showed striking similarities at distinct regions. The F‐actin cross‐linking molecule α‐actinin carries two characteristic EF‐hand structures highly homologous to the Ca2+‐binding loops of proteins from the calmodulin superfamily. An N‐terminal region that is conserved in α‐actinin from D. discoideum and vertebrates is also related to parts of the dystrophin sequence and might represent the F‐actin binding site. Severin, gelsolin, villin, and fragmin share homologous sequences that are believed to participate in the severing activity of these proteins.
Inactivation of the α‐actinin gene in Dictyostelium
Authors: Noegel Angelika A. , Witke Walter
Published: May 2005

α‐Actinin‐negative transformants of Dictyostelium have been obtained by transforming cells with a transformation vector carrying part of the α‐actinin gene in either sense or antisense orientation. The transformants did not produce detectable α‐actinin anymore and contained an altered RNA lacking the 3′ part of the coding sequences. The deficiency in α‐actinin was due to an integration of the transformation vector into the gene, since it could be detected by Southern blot analysis in the endogenous gene.
Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum
Authors: Boose Jeri Ann , Ziska Suzanne E. , Henderson Ellen J.
Published: May 2005

In order to identify the biological roles of protein‐linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature‐sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA‐resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA‐resistant cohesion, and reverted almost totally to wild‐type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA‐resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N‐linked oligosaccharides in aggregation‐stage intercellular cohesion.
Signals controlling cell differentiation and pattern formation in Dictyostelium
Authors: Kay Robert R. , Berks Mary , Traynor David , Taylor Graham W. , Masento Mark S. , Morris Howard R.
Published: May 2005

The major inducers of cell differentiation in Dictyostelium appear to be cyclic AMP and DIF‐1. Recently we have chemically identified DIF‐1, together with the closely related DIF‐2 and ‐3. They represent a new chemical class of potent effector molecules, based on a phenyl alkanone with chloro, hydroxy, and methoxy substitution of the benzene ring. Previous work has shown that DIF‐1 can induce prestalk‐specific gene expression within 15 min, whereas it suppresses prespore differentiation. Hence, DIF‐1 can control the choice of pathway of cell differentiation in Dictyostelium and is therefore likely to be involved in establishing the prestalk/prespore pattern in the aggregate. In support of this, we show that DIF treatment of slugs results in an enlarged prestalk zone. Cyclic AMP seems less likely to have such a pathway‐specifie role, but later in development it becomes inhibitory to stalk cell differentiation. This inhibition may be important in suppressing terminal stalk cell differentiation until culmination.

Spore differentiation can be induced efficiently by high levels of Br‐cyclic AMP, a permeant analogue of cyclic AMP. In this, it phenocopies certain spore‐maturation mutants, and we propose that during normal development spore differentiation is triggered by an elevation in intracellular cyclic AMP levels. How this elevation in cyclic AMP levels is brought about is not known. The experiments with Br‐cyclic AMP also provide the first direct evidence that elevated levels of intracellular cyclic AMP induce differentiation in Dictyostelium.
Lithium respecifiescyclic ‐AMP‐lnduced cell‐type specific gene expression in Dictyostelium
Authors: Van Lookeren Campagne Michiel M. , Wang Mei , Spek Wouter , Peters Dorien , Schaap Pauline
Published: May 2005

We investigated the effect of LiCl on pattern formation and cAMP‐regulated gene expression in Dictyostelium discoideum. In intact slugs, 5 mM LiCl induces an almost complete redifferentiation of prespore into prestalk cells. We found that LiCl acts by interfering with the transduction of extracellular cAMP to cell‐type‐specific gene expression; LiCl inhibits the induction of prespore‐specific gene expression by cAMP, while it promotes the induction of prestalk‐associated gene expression by cAMP. Our results indicate that two divergent pathways transduce the extracellular cAMP signal to, respectively, prestalk and prespore gene expression.
Geometry and spatial patterns in Polysphondylium pallidum
Authors: McNally J. G. , Cox E. C.
Published: May 2005

The formation of secondary sori in whorls of Polysphondylium pallidum provides an attractive model system for the study of symmetry breaking during morphogenesis. Tip‐specific antibodies that permit detection of very early stages in this patterning process are available. We have found that the patterns of tip‐specific antigen expression vary considerably depending on the size, shape, and developmental stage of the whorl. All of these patterns, however, are well explained by patterning models that rely on short‐range autocatalysis and long‐range inhibition, as exemplified by reaction‐diffusion theories. In the context of reaction‐diffusion, we discuss the possible effects of initial conditions, boundary conditions, and nonlinearities on the selection of patterns in P. pallidum whorls.
Euchromatic inversions causing mosaic gene expression in Drosophila hydei: A study of forked and Zebra
Authors: van Breugel Frans M. A.
Published: May 2005

In D. hydei two new mutants, In(1)f3 and IN(5)Z, show obvious mosaic gene expression. Their phenotypic expression is susceptible to the breeding temperature and to the addition of a supernumerary Y chromosome to the chromosome set. In this respect the mutants resemble standard cases of position‐effect variegation based on the action of heterochromatin. However, since neither centromeric nor sex chromosomal heterochromatin apparently are involved, the mutations point to a new type of variegation provoked by euchromatic sections. The mosaic patterns of these mutants, in particular those of In(1)f3, will be described.
Identification and expression of the gap segmentation gene hunchback in drosophila melanogaster
Authors: Bender Michael , Horikami Sandra , Cribbs David , Kaufman Thomas C.
Published: May 2005

Genetic analysis has shown that the gap segmentation gene hunchback (hb) is a member of the genetic hierarchy involved in pattern formation in Drosophila. To identify the hb gene, we have mapped the position of hb mutant breakpoints within a chromosomal walk of the 85A region by genomic Southern blots and determined the transcription pattern of DNA from the walk. We detect a single gene within the domain defined by breakpoint mapping. We conclude that we have identified the hunchback gene because three mutations that inactivate hb physically interrupt or delete this gene. Northern analysis shows that the hb gene gives rise to at least five overlapping transcripts ranging in length from 2.6 to 3.5 kilobases. S1 nuclease and primer extension experiments demonstrate that the gene employs two promoters and three polyadenylation sites. The two hb promoters have different temporal specificities. Transcripts arising from the upstream promoter are detected from 0–12 hours of embryogenesis as well as in adult female and male RNA preparations. Transcripts arising from the downstream promoter accumulate only from 0–6 hours of embryogenesis. During the syncytial blastoderm stage, transcripts from the hb gene accumulate over a broad anterior and a narrow posterior domain. This pattern sharpens during the late blastoderm/early gastrula stage to produce an embryo with two stripes of hybridization anterior and one stripe posterior. Later, hb transcripts are detected within the ventral hypoderm in extended germ band stage embryos.
Genetic determination of sporangiophore development in Phycomyces
Authors: Gutiérrez‐Corona Félix , Cerdé‐Olmedo Enrique
Published: May 2005

The mycelium of the fungus Phycomyces. essentially a giant multinucleate cell, produces two kinds of asexual reproductive structures, called macrophores and microphores, and a succession of structures for sexual reproduction. Following the treatment of spores with N‐methyl‐N′ ‐nitro‐N‐nitrosoguanidine, conditional imb mutants have been isolated that form no macrophores at 26°C, but do at 14°C. At the restrictive temperature, few imb mutants (2 of 13) develop microphores, and none is able to complete the sexual cycle. This suggests that genes responsible for macrophorogenesis are involved in microphorogenesis and in sexual development as well. Light reduces macrophorogenesis and totally abolishes microphorogenesis in the wild type under the conditions of our experiments. These photomorphogenetic effects require the normal function of genes madA and madB, which are responsible for phototropism. Light inhibits microphorogenesis in the two imb mutants that form microphores at the restrictive temperature. Genetic alterations of carotenogenesis lead to an excess of microphores and a scarcity of macrophores in the dark, but they have little influence on vegetative reproduction in the light.
Epithelial differentiation in Drosophila pupae
Authors: Mitchell Hershel K. , Petersen Nancy S.
Published: May 2005

The construction of cell hairs on the wings in developing pupae of Drosophila provides a unique system for studies of the regulation of differentiation in the absence of cell division. Early steps in hair construction are the extrusion of cell hairs and the deposition of the external impervious layer called “cuticulin.” Some properties of six of the most abundant proteins that are present during the early stages of hair construction are described. These proteins make up about 40% of the total protein of the preparation.
Genetic analysis of chorion formation in Drosophila melanogaster: I. The effects of one somatic‐specific and seven germ‐line‐specific mutations
Authors: Galanopoulos Vasilis K. , Orr William , Szabad János , Kafatos Fotis C.
Published: May 2005

Eight X‐linked recessive female sterile mutations, derived from a hybrid dysgenic screen of Drosophila melanogaster and representing eight distinct loci, have been characterized by genetic and ultrastructural analysis. Four have abnormal respiratory appendages, three have essentially normal appendages but show moderate defects in the endochorion, and one mutant, fs(1)nela, exhibits major defects in both the endochorion and the respiratory appendages. Germ line clones of all eight mutants were generated using the dominant female sterile technique. Seven of the eight mutations are germ line specific, indicating that, although the eggshell is produced by the follicular cells, germ line functions play a significant role in its elaboration. The mutant that shows major defects, fs(1)nela, is somatic line specific, and exerts its effect in the ovary.
Genetic analysis of modifiers affecting sexual dimorphism and temperature sensitivity of bx1 in Drosophila melanogaster
Authors: Larsen Ellen
Published: May 2005

Two modifiers of bithorax1 phenotypic expression are described. An X‐chromosome region is associated with sexual dimorphism in bx1 penetrance. It is hypothesized that sexual dimorphism is in part due to a lack of dosage compensation of the modifier, in males. A third chromosome region that segregates with the pink peach allele is implicated in mediating temperature sensitivity. By appropriate combinations of modifiers, both sexual dimorphism and temperature sensitivity can be greatly reduced.
A genetic switch, based on negative regulation, sharpens stripes in Drosophila embryos
Authors: Edgar Bruce A. , Odell Garrett M. , Schubiger Gerold
Published: May 2005

The pair‐rule genes hairy, runt, even‐skipped, and fushi tarazu express their mRNAs and proteins in striped patterns in the Drosophila embryo at the blastoderm stage. Previous studies have shown that the generation of these patterns depends upon products of the gap genes and upon interactions between the pair‐rule genes themselves. Here we show that blocking protein synthesis induces expression of each of the pair‐rule mRNAs in virtually all regions of the embryo. Our observations together with genetic studies carried out in other laboratories suggest that negative feedback between the pair‐rule genes plays a key role in striped expression of pair‐rule genes. We propose that stable proteins, present in all regions of the embryo, first activate transcription ofthese pair‐rule genes constitutively. Then, various combinations of unstable proteins repress their transcription in a patterned fashion; each stripe of accumulated products of a given pair‐rule gene marks a region where it was not repressed. We develop this idea in mathematical form and demonstrate that a network of mutual repression by pair‐rule genes can make each blastoderm nucleus into a genetic switch with two stable states. If preexisting gap gene patterns provide initial bias to the blastoderm nuclei, then the “bistable switch behavior” of the nuclei can refine an initially weak spatial bias into a final pattern of sharp stripes.
Cloning and dosage compensation of the 6‐phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster
Authors: Gutierrez Anthony G. , Christensen Alan C. , Manning Jerry E. , Lucchesi John C.
Published: May 2005

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X‐linked gene Pgd+, which encodes the enzyme 6‐phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd− phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.
Analysis of Adh gene regulation in Drosophila: Studies using somatic transformation
Authors: Shen Nancy L. L. , Subrahmanyam Guttina , Clark Wendy , Martin Presley , Sofer William
Published: May 2005

We have used in vitro mutagenesis and somatic transformation [Sofer and Martin, 1987a; Martin et al., 1986] to investigate the role of cis‐acting sequences in the control of alcohol dehydrogenase gene expression in larvae of Drosophila melanogaster. Two sets of experiments were carried out. In the first, a series of aeletions were constructed in the region upstream of the proximal transcriptional start site. In the second, one or both introns were removed from within the structural gene. These constructs (on circular plasmids) were injected into Adh‐null embryos and ADH activity was assayed in third instar larvae of the injected generation. The first set of experiments indicated that there are at least three distinct regulatory regions essential for larval activity located in the 5′ flanking region of the gene. One, in an area that includes the TATA box, was found to be necessary but not sufficient for larval ADH activity. Two others, further upstream, seemed to have enhancer‐like properties because their absence could be compensated by a second copy of the Adh gene on the same plasmid molecule. The second set of experiments showed that neither the tis‐sue distribution nor amount of ADH activity was affected by the removal of one or both introns from the Adh gene.
Larval fat body‐specific gene expression in D. melanogaster
Authors: Deutsch Jean , Laval Monique , Lepesant Jean‐Antoine , Maschat Florence , Pourrain Francine , Rat Luce
Published: May 2005

The P1 gene, together with the LSP‐1a, ‐1β, and ‐ly, LSP‐2, and P6 genes, is expressed exclusively in the larval fat body of D. melanogaster during the third instar. In vivo mapping of the cis‐acting regulatory sequences of the P1 gene was carried out using hybrid constructs with three different reporter genes and a combination of transient and germline transformation assays. This revealed that regulatory elements involved in the setting up of the temporal and spatial specificities of transcription of the P1 gene are located in a short DNA region immediately upstream of the mRNA transcription start. This region includes on element that behaves as a fat‐body transcriptional enhancer and element(s) required for ecdysone inducibility of transcription of the P1 gene.
Molecular genetics of dopa decarboxylase and biogenic amines in Drosophila
Authors: Hirsh J.
Published: May 2005

The gene Ddc encodes two isoforms of the enzyme dopa decarboxylase in Drosophila. These gene products catalyze the final steps in the synthesis of the biogenic amines sero‐tonin and dopamine. This article summarizes recent progress in understanding the tissue‐ and cell‐specific regulation of Ddc, which occurs at both the transcription and alternate splicing levels. In addition, results that are pertinent to understanding the roles of biogenic amines in the neurophysiology of Drosophila are discussed.
Molecular genetics ofDelta, a locus required for ectodermal differentiation inDrosophila
Authors: Alton Althea K. , Fechtel Kim , Kopczynski Casey C. , Shepard Scott B. , Kooh Pamela J. , Muskavitch Marc A. T.
Published: May 2005

Delta(Dl) is one of the six known zygotic neurogenic genes, each of which is essential for proper segregation of the embryonic ectoderm into neural and epidermal lineages. Molecular analysis ofDlreveals that it is a transcriptionally complex locus that yields multiple maternal and zygotic transcripts. DNA sequence analysis suggests that the predominant product of the locus is a putative transmembrane protein exhibiting homology to blood coagulation factors and epidermal growth factor of vertebrates. The structure of this product is consistent with the hypothesis thatDlparticipates in cell‐cell interactions that are central to establishment of the epidermal lineage within the developing ectoderm. Genetic analyses demonstrate thatDlmutations can modify the imaginal phenotypes that result from heterozygosity forNotch (N)mutations as well as the interaction between particular alleles ofNotch (N)andEnhancer of split [E(spl)]two other members of the neurogenic gene set. Vital interactions also occur betweenDlandN.Given the structures of products encoded byN,Dl, andE(spl), we suggest that the synergistic phenotypic interactions observed among mutations in these three loci result from physical, as opposed to regulatory, interactions.
Hemin increases production of β‐like globin RNA transcripts in human erythroleukemia K‐562 cells
Authors: Tsiftsoglou Asterios S. , Wong Willie , Robinson Stephen H. , Hensold Jack
Published: May 2005

Previous studies have indicated that control and hemin‐treated human eryth‐roleukemia K‐562 cells fail to produce adult‐type β‐globin mRNA transcripts and to translate them into nascent β‐globin chains. Expression of the β‐globin DNA sequences in K‐562 cells can occur, however, under certain conditions. To readdress this issue and to examine the possibility of whether these cells produce immature and untranslatable β‐globin RNA transcripts, we prepared total cyto‐plasmic RNA from control and inducer‐treated cells and performed Northern blot hybridization analysis using 5′ end‐labeled fragments of the human β‐globin DNA rather than 3′ end fragments as probes. Although hybridization of both cytoplasmic and nuclear K‐562 RNA with a32P‐labeled 3′ end fragment (1.6kb Bam H1 cut) coding for a large part of the first exon of β‐globin failed to detect β‐globin RNA transcripts, hybridization with a 5′ end 32P‐labeled 2.0kb Bam H1 fragment (coding for the third exon and part of the second) revealed the presence of relatively small (<7S) RNA molecules both in nuclear and cytoplasmic fraction. S1 nuclease mapping of both cytoplasmic and nuclear RNA with the use of 5′ end‐labeled 2.0 kb Bam H1 fragment of human β‐globin DNA indicated protection of a small portion located 64bp 5′ upstream from the Bam H1 site of the second exon. The amount of protected portion was relatively higher in K‐562 cells undergoing erythroid maturation. These findings suggest that control and differentiating K‐562 cells synthesize β‐globin‐like RNA transcripts that are 3′ end short, immature, and unable to give rise to adult β‐globin chains. These results also indicate that K‐562 cells may lack factors that are unique for transcription and processing of the human β‐globin RNA transcripts.
Heat‐shock proteins during pollen development in maize
Authors: Frova Carla , Taramino Graziana , Binelli Giorgio
Published: May 2005

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat‐shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat‐shock responses. One‐dimensional Sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis technique (SDS‐PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat‐shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat‐stress conditions; however, the consti‐tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.
Hyperinsulinemia in transgenic mice carrying multiple copies of the human insulin gene
Authors: Marban S. L. , Deloia J. A. , Gearhart J. D.
Published: May 2005

We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5′ human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C‐peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.
Developmental effects of modifiers of the vg mutant in Drosophila melanogaster
Authors: Cavicchi Sandro , Guerra Daniela , Natali Vanna , Giorgi Gianfranco , Pezzoli Cristina , Villani Luigi
Published: May 2005

An analysis of the modifiers affecting the expression of the vg gene was performed. We selected for weak and strong expression of the vg mutant in 2 segregating populations obtained by crossing a vestigial stock with an Oregon laboratory stock (O) and with a wild strain (B) captured near Bologna, Italy. The selection for enlarged wings was more effective in the vg B population where wild wings appeared from the lCth generation. The assay of the three major chromosomes showed that the modifiers are located on chromosomes 2 and 3. The mutant imaginal disc cell death phenotype is evident in vg/vg strains that have a wild‐type wing phenotype. It is suggested that the selected modifiers do not prevent cell death but induce regenerative growth.
Patterns of developmental and heritable change in methylation of the Suppressor‐mutator transposable element
Authors: Banks Jo Ann , Fedoroff Nina
Published: May 2005

Genetic inactivation of the Suppressor‐mutator (Spm) element is correlated with methylation of sequences surrounding the element's transcription initiation site. Several stages in the development of the plant have been identified during which element methylation is reproducibly altered. Loss of element methylation occurs during development of the embryo and early in vegetative growth of the tiller. Element methylation increases during vegetative growth and during development of male and female inflorescences. The susceptibility of element methylation to change during development correlates with the genetic stability of the element's phase of activity. Increases in methylation of sites both upstream and downstream of the Spm element's transcription initiation site parallel increases in the genetic stability of the inactive phase. These results strengthen the likelihood that methylation of C residues within specific regions of the element is important in maintaining the element in an inactive phase and is a component of the molecular mechanism that regulates element expression in plant development.
Transposable element‐induced mutations of the viviparous‐1 gene in maize
Authors: McCarty Donald R. , Carson Christian B. , Lazar Mark , Simonds S. Caprice
Published: May 2005

The viviparous‐1 (vp1) locus in maize is a developmental gene that controls diverse aspects of the maturation phase of seed development. Mutations of vp1 alter embryo sensitivity to the hormone abscisic acid and block formation of anthocyanin pigment. Molecular cloning of a Robertson Mutator‐induced mutant allele, vp1‐mum‐1, by transposable element tagging has allowed analysis of several transposon‐induced vp1 mutants. In the vp1‐Mc mutation, the gene is disrupted by 4.0 kbp insertion, which results in expression of a 3′ truncated mRNA. Phenotypically, this allele is at least partially functional in causing embryo dormancy, but is ineffective in controlling anthocyanin expression. This result suggests that disruption of the C‐terminal domain of the Vp1 protein specifically affects regulation of the anthocyanin pathway. A second Mutator‐ derived allele, vp1‐mum2, exhibits an unusual form of somatic mutability in which endosperm cells revert from wild‐type vp1 expression to a mutant condition. The vp1‐mum2 allele contains a 1.5 kbp Insertion that has no detectable homology to known Mu elements. This element is retained In wild‐type germinal revertants derived from vp1‐mum2 An apparent DNA modification affecting cleavage at an internal Sstl restriction site in the element correlates with vp1‐mum2 states that exhibit wild‐type Vp1 expression. A model involving mitotic assortment of modified and unmodified DNA strands during development is proposed for vp1‐mum2 somatic mutation.
Developmental and genetic aspects of Mutator excision in maize
Authors: Levy Avraham A. , Britt Anne Bagg , Luehrsen Kenneth R. , Chandler Vicki L. , Warren Christine , Walbot Virginia
Published: May 2005

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision.

Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.
w4‐Mutable line in soybean
Authors: Palmer R. G. , Hedges B. R. , Benavente R. S. , Groose R. W.
Published: May 2005

An unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4‐mutable line. Most plants in the line have chimeric flowers with purple sectors on a near‐white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype.

Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll‐deficient mutants found (CD‐1, CD‐2, and CD‐3) are inherited as single‐gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll‐deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD‐1, CD‐2, and CD‐3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.
Gene tagging in Petunia hybrida using homologous and heterologous transposable elements
Authors: Gerats Anton G. M. , Beld Marcel , Huits Henk , Prescott Andy
Published: May 2005

In this paper we describe the current state of knowledge of transposable element systems in Petunia hybrida. The main features of the unstable An1 alleles are discussed. The data on derivative (un)stable alleles at different loci are summarized. A simple strategy is outlined for random versus directed gene tagging using endogenous and heterologous elements. The progress in the cloning of endogenous elements is presented.
Temporal and spatial expression of a cytoskeletal actin gene in the ascidian Styela clava
Authors: Beach Rebecca L. , Jeffery William R.
Published: May 2005

We have cloned and characterized the temporal and spatial expression of ScCAl5, a cDNA clone encoding an actin gene in the ascidian Styela clava. The partial nucleotide and derived amino acid sequences of this single‐ copy gene suggest that it is a cytoskeletal actin. Northern analysis shows that ScCAl5 corresponds to a 1.8‐kb mRNA that is transcribed during oogenesis, during embryonic development, and in the adult. In situ hybridization shows that maternal ScCA15 mRNA is distributed uniformly in the cyto‐ plasm of the oocyte and unfertilized egg. During the period of ooplasmic segregation following fertilization, however, ScCAl5 mRNA appears to be translocated into the ectoplasm, a specialized cytoplasmic region of the egg. During the early cleavages, the ectoplasmic transcripts are partitioned to ectodermal cells in the animal hemisphere, which are precursors of the epidermis and nervous system of the larva. Maternal ScCA15 mRNA is degraded just before gastrulation and replaced by zygotic transcripts which begin to accumulate between the neurula and mid‐tailbud stages. Zygotic ScCAl5 mRNA accumulates primarily in the epidermal and neural cells, although lower levels of these transcripts may also be present in tail muscle cells. These results show that two mechanisms are used to concentrate ScCA15mRNA in the ectodermal cells during development: (1) localization and differential segregation of maternal transcripts and (2) specific expression of the ScCA15 gene. ScCAl5 mRNA is detected by in situ hybridization in the testes, ovaries, alimentary tract, and endostyle of adults. In the testes, ScCA15 mRNA is present in developing sperm, whereas in the ovary, these transcripts are present in the germinal epithelium and developing oocytes. In the alimentary tract, ScCAl5 mRNA is confined to the gastric epithelium of the esophagus, stomach, and intestine. Since the ScCA15 gene is expressed in embryonic and adult tissues that are undergoing rapid cell division, this actin is likely to function in some aspect of cell proliferation.