Suppression of flavonoid flower pigmentation genes in Petunia hybrida by the introduction of antisense and sense genes.
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Acyltransferases↗
Biomedical subjects
J M Kooter
Publications and source records attributed to J M Kooter.
Conditional inhibition of beta-glucuronidase expression by antisense gene fragments in petunia protoplasts.
Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of beta-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60-70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.
Base Sequence↗
The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme.
The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.
Amino Acid Sequence↗
Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.
Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Acyltransferases↗
Boundaries of telomere conversion in Trypanosoma brucei.
Active genes for variant-specific surface glycoproteins (VSGs) reside in telomeric expression sites and may be replaced by other VSG genes via telomere conversions. The availability of a complete map of expression site 221 in variant 221a made it possible to determine the boundaries of such conversions and the sequences that are involved. We have analysed five trypanosome populations that arose from variant 221a through replacement of the 221 gene by another VSG gene. In each of these relapsed populations the telomere conversion ends at a different position in the expression site. In the relapsed population, 221aR3, the boundary was found in the coding region of an expression-site-associated gene (ESAG). This ESAG-2 codes for a potential 368-aa protein of unknown function; it contains a N-terminal signal peptide for mediating transfer to the endoplasmic reticulum and six potential N-glycosylation sites. It shares these structural features with the ESAG-1 protein encoded in the same expression site. ESAG-2 is a member of a large gene family which includes non-functional genes. In 221aR3, the partial conversion of ESAG-2 by an ESAG-2-like sequence has disrupted the open reading frame. The two ESAG-2 sequences are similar (92% identity) suggesting that sequence homology between telomeres provides the opportunity for gene conversion.
Amino Acid Sequence↗
The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei.
The variant specific surface glycoprotein (VSG) genes of T. brucei are expressed in telomeric expression sites. We have determined the structure of the active site in trypanosome variant 221a, which contains VSG gene 221, by analysis of cloned DNA segments that represent 65 kb of the 5'-flanking region of the VSG gene. In nuclear run-on experiments, 57 kb of adjacent sequences are cotranscribed with the VSG gene at approximately similar rates and in the alpha-amanitin-resistant manner characteristic of VSG genes. Besides the VSG mRNA, this expression site yields at least seven stable RNAs, suggesting that it is a multicistronic transcription unit. Our results also show that insertion of a transcriptional terminator is not the general mechanism of switching off expression sites.
Animals↗
Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene.
We have used inactivation of transcription by UV irradiation to map transcription units in trypanosomes. The relative inactivation rate of the transcription of mini-exon, 5S, and rRNA genes was inversely proportional to the previously estimated lengths of these transcription units. The telomeric transcription unit containing the gene for variant-specific surface glycoprotein (VSG) 221 was inactivated as a single unit of 60 kb. This long transcription unit comprises at least one other protein-coding gene and yields seven other stable mRNAs. These data thus provide evidence for a multicistronic transcription unit for cellular genes in a eukaryote.
Animals↗
Coincident multiple activations of the same surface antigen gene in Trypanosoma brucei.
Trypanosomes with a coat of variant surface glycoprotein (VSG) 118, consistently appear around day 20 when a rabbit is infected with Trypanosoma brucei strain 427. There is a single chromosome-internal gene for VSG 118 and this is activated by duplicative transposition to a telomeric expression site. We show here that the expression-linked extra copy of VSG gene 118 in a day 18 population of a chronic infection is heterogeneous, and we infer that the population is not monoclonal but is the result of multiple independent activations of the 118 gene. We show that the heterogeneity of expression-linked extra copies is also present in other trypanosome populations expressing chromosome-internal VSG genes. We present a model for the timing of VSG gene activation during chronic infection that emphasizes two features: the relative activation and inactivation frequencies of different expression sites, and the degree of homology of the sequences flanking VSG genes with expression sites.
Antigens, Protozoan↗
Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes.
Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.
Antigens, Protozoan↗
Transcription of a transposed trypanosome surface antigen gene starts upstream of the transposed segment.
The non-telomeric variant surface glycoprotein (VSG) genes in Trypanosoma brucei are activated by a duplicative transposition to a telomeric expression site. We have determined the 5' end of the transposed segment of the gene for VSG 117 and infer from comparison with similar data obtained by others that the crossover can occur at variable positions within short repeats present upstream of this gene and in the expression site. We have analysed nascent and steady state transcripts of the transposed gene and its neighbouring expression site DNA. The results indicate that transcription starts upstream of the transposed gene segment in the expression site and that transcripts are rapidly processed at specific points identified by protection of DNA-RNA hybrids against digestion by nuclease S1 or Exo VII. Hence, this gene appears to be activated by a process akin to promoter addition.
Animals↗
Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis.
Mature mRNAs of Trypanosoma brucei have a common 5' terminal sequence of 35 nucleotides. This is acquired by an unknown mechanism from the 5' end of a separately transcribed precursor RNA of about 140 nt called the mini-exon-derived RNA or medRNA. We have investigated the nature of the 5' ends of mature mRNAs and of the medRNA by chemical decapping and enzymic recapping. We infer that a 5' cap is present on both of these RNAs and conclude that the mini-exon-derived RNA donates its 5' cap along with the mini-exon sequence to the pre-mRNA. Using nuclear run-on experiments we show that medRNA synthesis is much more sensitive to alpha-amanitin than 5S RNA synthesis and only slightly less sensitive than tubulin gene transcription. This result, together with the presence of a cap at the 5' end of the medRNA indicates that the mini-exon is transcribed by an RNA polymerase II type enzyme. Our experiments also confirm the existence of a second minor medRNA of about 125 nt and show the presence of other small capped RNAs possibly analogous to the small nuclear RNAs of other organisms.
Amanitins↗
Two simultaneously active VSG gene transcription units in a single Trypanosoma brucei variant.
Trypanosomes can change their surface coat either by slotting a different surface antigen gene copy into an active (telomeric) expression site or by activating a new VSG gene expression site and inactivating the old one. How expression sites are activated or inactivated is not clear. We report an exceptional trypanosome variant in which the inactivation of a surface antigen gene is accompanied by a 30 kb DNA insertion 5' of the gene. Transcription of the region upstream of the insertion continues unaltered and retains the characteristic insensitivity to alpha-amanitin of VSG gene transcription units, showing that the expression site is still active. The expressed VSG gene in this trypanosome variant resides in another telomere. Hence, two VSG gene transcription units can be simultaneously active. This argues against a single mobile activating element controlling VSG gene transcription and favors a stochastic model of telomere activation/inactivation.
Animals↗
Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.
The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.
Animals↗
Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes.
Many, if not all, mRNAs in T.brucei start with the same sequence of 35 nucleotides, separately encoded in clustered so-called mini-exon repeats. From these mini-exon repeats a 141-nt precursor RNA with the 35-nt sequence at its 5' end is transcribed. Indirect evidence suggests that this RNA is linked in a second step to pre-mRNA transcripts. We have studied the sensitivity of RNA synthesis to alpha-amanitin in isolated trypanosome nuclei. Transcription of several protein coding genes is almost completely inhibited by a concentration of 5 micrograms alpha-amanitin per ml, whereas strong inhibition of mini-exon transcription is achieved with 200 micrograms alpha-amanitin per ml. In contrast, transcription of genes for variant surface glycoproteins (VSGs) is not inhibited by 1000 micrograms alpha-amanitin per ml, as is transcription of the genes for the major rRNAs. Since the mature VSG mRNAs start with the 35-nt sequence, our results provide additional evidence that the 35-nt sequence and the main part of VSG mRNA are produced from independent transcription units, these are transcribed by (partly) different RNA polymerases.
Amanitins↗
Discontinuous synthesis of mRNA in trypanosomes.
Many trypanosome mRNAs have the same sequence of 35 nucleotides at their 5' end, encoded by a mini-exon located in 1.35-kb tandemly linked repeats. We have analysed nascent and steady-state mini-exon transcripts to determine how the mini-exon sequence is joined to the main part of trypanosome mRNAs. We show here that steady-state RNA from Trypanosoma brucei contains a transcript of 141 nucleotides that starts at the 5' border of the mini-exon. Isolated nuclei transcribe the segment corresponding to the 141 nucleotide RNA at a high rate; transcription of other areas of the 1.35-kb mini-exon repeat is approximately 750-fold lower. We propose that transcription of protein-coding genes in trypanosomes is discontinuous and involves the 141-nucleotide transcript as an intermediate.
Animals↗
The control of variant surface antigen synthesis in trypanosomes.
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Animals↗
Telomere conversion in trypanosomes.
Activation of the gene coding for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei proceeds via a duplicative transposition to a telomeric expression site. The resulting active expression-linked extra copy (ELC) is usually flanked by DNA that lacks sites for most restriction enzymes and that is thought to interfere with the cloning of the ELC as recombinant DNA in Escherichia coli. We have circumvented this problem by cloning an aberrant 118 ELC gene, flanked at the 3'-side by at least 1 kb DNA, that contains restriction enzyme sites. Our analysis shows that this DNA and the 3'-end of the 118 ELC gene are derived from another VSG gene (1.1006) that is permanently located at a telomeric position. We propose that the 3'-end of the 1.1006 gene and (all of) its 3' flanking sequence moved to the expression site by a telomere conversion. Such a telomere conversion can also account for the appearance of an extra copy of the 1.1006 gene detected in a sub-population of our trypanosome strain.
Amino Acid Sequence↗
The detection and use of hemoglobin mutants in the direct analysis of human globin genes.
Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma-, and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.
Amino Acid Sequence↗