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U Wobus

Publications and source records attributed to U Wobus.

At least 37 records · Page 2Linked to original sources
PubMed1995

Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression.

We have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated.

Base Sequence↗
PubMed1994

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes.

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamine/glutamic acid-rich region encoded by the C-terminal part of the alpha chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342-350) and limited own data.

Amino Acid Sequence↗
PubMed1994

Tissue-specific expression of an oat 12S seed globulin gene in developing tobacco seeds: differential mRNA and protein accumulation.

We studied the expression of the oat globulin gene asglo5 in developing transgenic tobacco seeds. The asglo5 gene promoter directed transcription in the endosperm as well as in the provascular tissue, the presumptive root tip and the shoot apical meristem of the embryo as revealed by GUS reporter gene constructs and in situ hybridization. However, immunological tissue printing detected the oat protein exclusively in the tobacco endosperm, suggesting that extensive post-transcriptional regulatory processes influence the expression of the monocot transgene in the dicot host.

Allergens↗
PubMed1993

A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene.

We have identified cis-regulatory elements within the 5'-upstream region of a Vicia faba non-storage seed protein gene, called usp, by studying the expression of usp-promoter deletion fragments fused to reporter genes in transgenic tobacco seeds. 0.4 kb of usp upstream sequence contain at least six, but probably more, distinct cis-regulatory elements which are responsible for seemingly all quantitative, spatial and temporal aspects of expression. Expression-increasing and -decreasing elements are interspersed and include an AT-rich sequence, a G-box element and a CATGCATG motif. The latter acts as a negative element in contrast to what has been found for the same motif in legumin- and vicilin-type seed storage protein gene promoters. Seed specificity of expression is mainly determined by the -68/+51 region which confers, however, only very low levels of expression. The data support the combinatorial model of promoter function.

Base Sequence↗
PubMed1993

A sucrose-synthase gene of Vicia faba L.: expression pattern in developing seeds in relation to starch synthesis and metabolic regulation.

Copy-DNA clones encoding a single class of sucrose-synthase (SUCS; EC 2.4.1.13) subunit have been isolated and sequenced from a Vicia faba L. seed cotyledonary library. Southern analyses indicated the existence of only one gene. Transcript levels determined by Northern blot hybridisation steadily increased until the middle of development [25-35 days after flowering (DAF)] and declined thereafter. Sucrose levels approximately paralleled levels of SUCS mRNA. The activity of SUCS increased with decreasing fructose and glucose concentrations and peaked about 10 d later than mRNA levels. In-vitro culture experiments demonstrated that increasing the sucrose concentration leads to increased levels of SUCS mRNA. The SUCS mRNA was also synthesised in seed-coat tissue, but in lower amounts than in cotyledons and with a different developmental profile. The early peak level of SUCS mRNA (20 DAF) in seed coats coincided with the peak in the amount of sucrose and with a peak of transiently synthesised starch.

Amino Acid Sequence↗
PubMed1993

Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes.

A new technique is reported for the physical mapping of low copy DNA sequences on plant chromosomes. Individual chromosomes were microisolated and their DNA used as the target for the polymerase chain reaction in order to identify the chromosome carrying a specific gene sequence. The use of defined translocation chromosomes further refined the resolution of the method to a subchromosomal level. To demonstrate the applicability of the procedure genes have been localized coding for vicilin seed storage proteins on the field bean Vicia faba L. in a region which includes the centromere and the proximal parts of the short and the long arms of chromosome II.

Base Sequence↗
PubMed1992

Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue-specific expression of a legumin gene.

A 2.4 kb fragment containing the 5'-flanking region and the 5'-noncoding sequence of the Vicia faba legumin gene LeB4 mediates high level seed-specific expression in transgenic tobacco plants. Deleted derivatives of this legumin upstream sequence were fused to the npt-II reporter gene to determine the tissue-specific activity of the chimeric constructs in stably transformed tobacco plants. The results indicate the presence of positive regulatory, enhancer-like cis elements within 566 bp of the upstream sequence. Most importantly, however, these elements are only fully functional in conjunction with the core motif CATGCATG of the legumin box around position -95, since destruction of the motif by a 6 bp deletion in an otherwise intact 2.4 kb upstream sequence drastically reduces expression in seeds. At the same time, low level expression in leaves is observed. The occurrence of similar CATGCATG consensus cis elements with alternating purine and pyrimidine base pairs in front of several other plant genes suggests a functional role of the motif in a wider range of plant promoters.

Base Sequence↗
PubMed1991

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants.

We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5' flanking sequence and the total 5' untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase II and beta-glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.

Amino Acid Sequence↗
PubMed1991

Upstream sequences regulating legumin gene expression in heterologous transgenic plants.

We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.

Blotting, Southern↗
PubMed1990

A new transposable element in Chironomus thummi.

A 1.7 kb long transposable element called TECth1 was found in the 3' flanking region of a Chironomus thummi Balbiani ring gene. As shown by sequence comparison with a second copy, TECth1 is characterized by a perfect terminal inverted repeat of 17 bp flanked by a duplicated target site of 8 bp, four internal imperfect inverted repeats of 17 to 26 bp and terminal regions of about 0.25 kb with a high number of short direct repeats of the consensus sequence ACTTT or permutated and mutated forms such as TTTAC or ACTAT. The terminal inverted repeats and the 8 bp target site duplication are reminiscent of Drosophila P and hobo elements but no long open reading frame starting with ATG is present, suggesting that the two TECth1 copies studied represent deletion derivatives of a longer element coding for its own transposase. In situ hybridization revealed about 75 labelled sites distributed over all chromosomes with the Balbiani ring locus most strongly labelled. Fifty percent of the sites are specific for a given individual, and these variable sites are often heterozygous for the element.

Animals↗
PubMed1989

The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes.

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3' flanking sequences. On the other hand, two pseudogenes (psi LeB1, psi LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.

Amino Acid Sequence↗
PubMed1986

The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element.

The field bean, Vicia faba L. var. minor, possesses two sub-families of 11 S legumin genes named A and B. We isolated from a genomic library a B-type gene (LeB4) and determined its primary DNA sequence. Gene LeB4 codes for a 484 amino acid residue prepropolypeptide, encompassing a signal peptide of 22 amino acid residues, an acidic, very hydrophilic alpha-chain of 281 residues and a basic, somewhat hydrophobic beta-chain of 181 residues. The latter two coding regions are immediately contiguous, but each is interrupted by a short intron. Type A legumin genes from soybean and pea are known to have introns in the same two positions, in addition to an extra intron (within the alpha-coding sequence). Sequence comparisons of legumin genes from these three plants revealed a highly conserved sequence element of at least 28 bp, centered at approximately 100 bp upstream of each cap site. The element is absent from the equivalent position of all non-legumin and other plant and fungal genes examined. We tentatively name this element "legumin box" and suggest that it may have a function in the regulation of legumin gene expression.

Amino Acid Sequence↗
PubMed1986

The 3' ends of two genes in the Balbiani ring c locus of Chironomus thummi.

The 3'-end sequences of two nonallelic genes derived from the Balbiani ring c (BRc) locus of Chironomus thummi are described. Only one of the genes appears to be transcribed abundantly in normal late larval salivary glands. The two sequences are highly similar, even in the 3' untranslated regions, but sharply diverge beyond the polyadenylation site. Together with evidence from the 3' ends of BR1 and BR2 genes of C. pallidivittatus and C. tentans, independently characterized by others, this result suggests the existence of a sequence-homogenization mechanism that operates across the 3' ends of all BR genes characterized to date. The 3'-terminal coding region of each BRc gene is divided into two portions by a short intron. The upstream portion is homologous to and continuous with the tandem repeats that make up the internal core of each BR gene; however, that portion is variant in sequence relative to the core, and apparently is not subject to the homogenization process that operates on the core repeats. The portion downstream of the intron encodes a unique, 111-residue polypeptide highly different from the rest of the BRc product. The evolution of the various segments of the BRc genes is discussed.

Animals↗
PubMed1984

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane.

Hybridization-selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell-free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium-washed membranes (K-RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K-RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557-561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.

Animals↗
PubMed1984

Self-complementary DNA sequences within the BRc gene of Chironomus thummi.

The Balbiani ring c (BRc) DNA of Chironomus thummi consists of tandemly arranged 249-base pair (bp) repeats, which represent the major part of the gene (Bäumlein et al. 1982a) and are transcribed and translated in a periodic polypeptide of unusual size. To obtain further information on the DNA sequence organization of that gene a recombinant phage (lambda CthBRc-1) with a relatively long insert (containing predominantly 249-bp repeats) was studied by electron microscopy (EM). lambda CthBRc-1 was found to undergo specific sequence elimination of BRc DNA resulting in heterogeneous size distribution of insert length within the limits of the cloning capacity of the phage with a maximum around 15 kilobase pairs (kb). The EM analysis of R loops formed between recombinant molecules and poly(A)+RNA (containing the transcripts of BRc and BRb) revealed the existence of self-complementary inverted and direct repeats as further sequence elements of BRc DNA scattered throughout a long portion of the BRc transcription unit. Different intrastrand structures (stems, hairpins, complex loops) originate from the renaturation of several sets of self-complementary repeats. Most double-stranded regions fell into one main-size class with an average length of 0.1 kb. The overall data suggest that self-complementary repeats belong to the same DNA sequence family and are able to cooperate in the formation of loops of different size and complexity. The results are discussed in relation to the functional significance of self-complementary inverted repeats (palindromes) for BRc expression.

Animals↗