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J N Mol

Publications and source records attributed to J N Mol.

At least 37 records · Page 2Linked to original sources
PubMed1990

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.

Bacteria↗
PubMed1990

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants.

The activity, tissue specificity and temporal expression of the tandem promoter region preceding a maize zein gene (zE19, encoding a 19 kDa zein protein) were tested in transgenic Petunia plants. To simplify the analysis, the tandem promoter as well as each of the two separate promoter regions were fused to the beta-glucuronidase (GUS) reporter gene. All of the three constructs directed the synthesis of GUS in the endosperm of transformed seeds indicating that both separate promoters are independently activated and show the same tissue and cell type specificity observed for zein genes in maize. The kinetics of accumulation and the localization of GUS activity are not coordinated with those of Petunia endogenous seed storage proteins during the development of transformed seeds. Unexpectedly, we detected high levels of GUS activity in anthers of transformed Petunia plants for all three constructs. This appears to reflect the expression pattern of zein genes in maize, since we detect zein transcripts in anthers. Finally, we discuss the possible origin and function of the tandem promoter arrangement on the basis of these data.

Base Sequence↗
PubMed1990

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression.

In order to scan the 5' flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5' end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from -67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from -142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (-59 and -52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.

Acyltransferases↗
PubMed1990

Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect.

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene. Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3' half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5' half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes. Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.

Acyltransferases↗
PubMed1990

Stamens and Gibberellic Acid in the Regulation of Flavonoid Gene Expression in the Corolla of Petunia hybrida.

Stamen removal at an early stage of flower development inhibits anthocyanin synthesis and chalcone flavanon isomerase (CHI) enzyme activity in corollas of Petunia hybrida. The inhibition can be overcome by gibberellic acid (GA(3)) application. Gibberellin also induces anthocyanin synthesis in detached, young green corollas, grown in vitro in a sucrose medium and promotes CHI enzyme activity. Western blot analysis indicates an increase in chalcone synthase (CHS) and CHI protein levels following GA(3) treatment in both the in vivo and the in vitro systems. Northern blot analysis shows a higher level of steady-state mRNAs for CHS and CHI 24 hours after GA(3) application. In corollas from a transgenic plant containing a beta-glucuronidase gene driven by a CHI promoter, a sixfold increase of beta-glucuronidase activity was measured following GA(3) application. The mode of action of stamens and GA(3) control over flavonoid gene expression is discussed.

Journal Article↗
PubMed1990

Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.

Acyltransferases↗
PubMed1990

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.

Base Sequence↗
PubMed1989

Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida.

Chalcone synthase-encoding genes (chs) in Petunia hybrida comprise a multigene family. Some of the chs genes have been grouped into a subfamily, based upon their strong cross-hybridization and tight genomic linkage. From genomic libraries eight 'complete' chs genes, two chs gene 5'-fragments and two chs gene 3'-fragments have been isolated. The nucleotide sequence of six complete chs genes is presented and discussed in relation to their evolutionary origin and expression in different tissues. Each member of the family consists of two exons separated by an intron of variable size and sequence, which is located at a conserved position. The chs gene fragments represent single exons. Homology between non-linked chs genes is approx. 80% at the DNA level and restricted to protein-coding sequences. Homology between subfamily members (which are tightly linked) is higher (90-99%) and extends into untranslated regions of the gene, strengthening the view that they arose by recent gene duplications. The chsD gene contains a mutated translation stop codon, suggesting that this is an inactive (pseudo)gene. None of the other members of the gene family exhibits characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must have been a recent event. Homology at the protein level between some (expressed) chs genes is surprisingly low. The possibility that these genes encode proteins with slightly different enzymatic activities is discussed.

Acyltransferases↗
PubMed1988

Antisense genes in plants: an overview.

Plants are the first multicellular higher eukaryotic organisms in which artificial antisense genes have been shown to down-regulate target gene expression. Manipulations with an antisense gene can serve as a tool to study the effect of a particular plant gene inactivation, the interaction of gene products whose genes are coordinately expressed, or the functional analysis of cryptic genes. Transgenic plants harbouring an antisense gene already gave rise to patentable new characteristics, showing that the technique has great scientific and economic value.

Genes↗
PubMed1988

Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes.

In this paper we report the isolation of cDNA clones encoding the flavonoid-biosynthetic enzyme chalcone flavanone isomerase (CHI) from Petunia hybrida. A nearly full size cDNA clone, isolated from a corolla-specific expression library, was characterized by sequence analysis. Using this CHI cDNA and the previously cloned flavonoid-specific chalcone synthase (CHS) cDNA we show that CHI and CHS genes are coordinately and tissue-specifically expressed in a developmental and light-regulated manner. Furthermore, coordinate induction of both mRNAs is observed after continuous irradiation of Petunia plantlets with UV light, probably as part of the plants UV defence mechanism. The two CHI genes, denoted A and B, were isolated from a genomic library of the Petunia inbred line V30. Both genes are transcriptionally active: gene A is transcribed in corolla, tube and UV-irradiated plantlets (1.0 kb mRNA), whereas gene B is only transcribed in immature anthers (1.0 kb mRNA). In combination with Southern blot analysis these data implicate the presence of two distinct non-allelic CHI genes in the genome of the P. hybrida line V30. Unexpectedly, mature anthers accumulate a 0.3 kb larger CHI RNA. This RNA is transcribed from CHI gene A and has a 0.3 kb 5' extension relative to the gene A transcript found in corolla tissue. Furthermore it is neither coordinately expressed with CHS mRNA nor UV inducible. Its biological function is still obscure, since no active CHI enzyme could be demonstrated in the same tissue.

Acyltransferases↗
PubMed1987

A novel purification procedure for chalcone flavanone isomerase from Petunia hybrida and the use of its antibodies to characterize the Po mutation.

We have purified chalcone flavanone isomerase (CHI) from flowerbuds of Petunia hybrida to high purity. We made use of an affinity matrix consisting of Sepharose-bound Dextran Blue that is known to bind proteins containing the dinucleotide fold [S. T. Thompson, K. H. Cass, and E. Stellwagen (1975) Proc. Natl. Acad. Sci. USA 72, 669-672]. The final step, consisting of preparative elution from a denaturing acrylamide gel, yielded an approximately 2000-fold purified CHI protein. The enzyme is a single polypeptide with Mr = 29,000, and highly specific antiserum was raised against it. Using this antiserum it was shown that corolla and anther tissues express different forms of the enzyme as judged by pI. Furthermore, the absence of immunoreactive CHI was demonstrated in a mutant of P. hybrida (genotype popo) which accumulates 2',4,4',6'-tetrahydroxy-chalcone in anthers as a consequence of lack of enzyme activity.

Animals↗
PubMed1986

Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family.

Twenty independent, petal-specific chalcone synthase (CHS) cDNA clones have been isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone in Southern blot analysis reveals the presence of multiple CHS gene copies in the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cDNA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptionally active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene.

Acyltransferases↗
PubMed1985

The induction of myeloid leukaemias by Rauscher murine leukaemia virus.

A number of cloned viral preparations isolated from Rauscher virus-producing JLS-V5 cells were compared in their competence to induce different types of leukaemias. All preparations were able to induce myeloid leukaemias, but the induction of lymphatic or erythroid leukaemias was also observed. Serial infection of newborn mice with either cell-free extracts or serum from animals suffering from a myeloid leukaemia did not result in the occurrence of relatively more myeloid leukaemias nor did the infection with virus harvested from ascites fluid of permanent myeloid cell lines. It appears that the mechanism by which myeloid leukaemias are induced is not virus-specific.

Animals↗
PubMed1982

Rauscher spleen focus-forming virus: biological properties and relationship to helper viruses.

A Rauscher virus (RV)-transformed erythroid cell line, RA-1, was shown to be a non-producer cell line. RA-1 cells express not only gp51-54 env-related glycoprotein, but also gp70, which is more closely related to gp51-54 coded by a recombinant env gene than to the MuLV-R gp70. RA-1 cells could be infected by Friend, Moloney and Gross viruses, but not by the homologous Rauscher murine leukaemia virus. Rescue of spleen focus-forming activity was obtained on infection of these cells with MuLV-F or MuLV-Mol, but not with MuLV-Gross. The RNA of the RV complex resembles closely that of Friend virus (FV). It contains a 32S, presumably defective, genome, which most likely is responsible for spleen focus formation, and a 35S helper virus genome. Oligonucleotide fingerprint data suggest that RV has evolved independently of FV. Erythroid early BFU-E cells of mice infected with RV of Friend helper virus-infected RA-1 cells were shown to require no addition of conditioned medium to form large erythroid colonies (BFU-E) in the presence of only small amounts of erythropoietin.

Animals↗
PubMed1981

The relationship between genomic RNAs of polycythaemic forms of spleen focus-forming Friend virus and its helper virus.

We have studied the relationship between Friend spleen focus-forming virus (SFFV) and its helper lymphoid leukaemia virus (LLV) by comparing RNase T1 fingerprints of genomic RNAs. Our data indicate that about 70% of the SFFV sequence is a perfect copy of parts of the helper genome. We conclude that our SFFV and LLV isolates have co-evolved very closely and that SFFV-specific sequences are not identical in different Friend virus isolates.

Base Sequence↗
PubMed1978

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA.

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.

Adenine↗