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Biomedical subjects

H Saedler

Publications and source records attributed to H Saedler.

At least 19 recordsLinked to original sources

Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development.

We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non-permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.

Alleles

Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS-box gene squamosa in Antirrhinum majus.

Anomalous flowering of the Antirrhinum majus mutant squamosa (squa) is characterized by excessive formation of bracts and the production of relatively few and often malformed or incomplete flowers. To study the function of squamosa in the commitment of an inflorescence lateral meristem to floral development, the gene was cloned and its genomic structure, a well as that of four mutant alleles, was determined. SQUA is a member of a family of transcription factors which contain the MADS-box, a conserved DNA binding domain. In addition, we analysed the temporal and spatial expression pattern of the squa gene. Low transcriptional activity of squa is detectable in bracts and in the leaves immediately below the inflorescence. High squa transcript levels are seen in the inflorescence lateral meristems as soon as they are formed in the axils of bracts. Squa transcriptional activity persists through later stages of floral morphogenesis, with the exception of stamen differentiation. Although necessary for shaping a normal racemose inflorescence, the squa function is not absolutely essential for flower development. We discuss the function of the gene during flowering, its likely functional redundancy and its possible interaction with other genes participating in the genetic control of flower formation in Antirrhinum.

Alleles

GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.

GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.

Alleles

Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype.

30,000 transgenic petunia plants carrying a single copy of the maize A1 gene, encoding a dihydroflavonol reductase, which confers a salmon red flower colour phenotype on the petunia plant, were grown in a field test. During the growing season plants with flowers deviating from this salmon red colour, such as those showing white or variegated phenotypes and plants with flowers exhibiting only weak pigmentation were observed with varying frequencies. While four white flowering plants were shown at the molecular level to be mutants in which part of the A1 gene had been deleted, other white flowering plants, as well as 13 representative plants tested out of a total of 57 variegated individuals were not mutants but rather showed hypermethylation of the 35S promoter directing A1 gene expression. This was in contrast to the homogeneous fully red flowering plants in which no methylation of the 35S promoter was observed. While blossoms on plants flowering early in the season were predominantly red, later flowers on the same plants showed weaker coloration. Once again the reduction of the A1-specific phenotype correlated with the methylation of the 35S promoter. This variation in coloration seems to be dependent not only on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made.

Alcohol Oxidoreductases

Molecular analysis of tap2, an anther-specific gene from Antirrhinum majus.

Deficiens is a floral homeotic gene of Antirrhinum majus, mutation of which results in transformation of petals to sepals and stamens to carpels. In a search for putative target genes, controlled by this regulatory locus, cDNA clones representing genes, that are expressed in wild type but not in the deficiens mutant flowers, were isolated by differential screening. The molecular structure and the expression pattern of one of these genes, tap2, is described. Tap2 is transiently and tissue-specifically expressed in the tapetum of the anthers. It encodes a 131 amino-acids-long protein with a hydrophobic N-terminus, displaying all characteristic features of a signal peptide. This indicates that the TAP2 protein may be secreted.

Amino Acid Sequence

The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene in.

Two chalcone synthase genes in maize have been cloned and molecularly characterized to be the C2 and the Whp (white pollen) locus. The two genes have highly homologous exon sequences but differ considerably in sequences 5' upstream and 3' downstream of the coding region, as well as in their introns. Northern and Western experiments of chalcone synthase expression in various tissues and in different genotypes indicated that C2 and Whp are differently regulated. The expression of Whp in maize aleurone is dependent on the presence of the recessive allele of the gene intensifier (in). The regulatory effect of in on Whp expression is not detectable at the transcriptional level, but seems to take place during translation.

Acyltransferases

Molecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild type, but not in the deficiens mutant of Antirrhinum majus.

Deficiens, a homeotic gene involved in the genetic control of flower development, codes for a putative transcription factor. Upon mutation of the gene, petals are transformed to sepals and stamens to carpels, indicating that deficiens is essential for the activation of genes required for petal and stamen formation. In a search for putative target genes of deficiens, several stamen- and petal-specific genes were cloned that are expressed in wild type but not in the deficiensglobifera mutant. In this report the molecular characterization of two of these genes, tap1 and fil1, is presented. They are transiently expressed during flower development. In situ hybridization data demonstrate that tap1 is expressed in the tapetum of the anthers and fil1 in the filament of the stamen and at the bases of the petals. Both genes encode small proteins with N-terminal hydrophobic domains suggesting that they are secreted. We discuss possible functions of the gene products and their relationship to the deficiens gene.

Amino Acid Sequence

The transposable element Tam1 from Antirrhinum majus shows structural homology to the maize transposon En/Spm and has no sequence specificity of insertion.

We present the genomic structure of Tam1, a transposable element from Antirrhinum majus. The Tam1 element is 15.2 kb long and includes two genes that are transcribed to produce a 2.4 kb (tnp1) and a 5 kb mRNA (tnp2). These transcripts partially overlap and the exons are scattered over the whole element. Tnp1 encodes a 53 kDa protein as deduced from the cDNA sequence. The 5 kb transcript of tnp2 contains an open reading frame that shares 45% homology with part of the tnpD gene of En/Spm from maize and 48% homology with an open reading frame of the Tgm element from Glycine max. We discuss the possible functions of these genes by analogy with En/Spm. Additionally, a number of flanking sequences of Tam1 insertions were analysed to investigate the sequence specificity of insertion. From these studies we conclude that Tam1 transposes predominantly into AT-rich regions that can be unique as well as repetitive. No specific target sequence of insertion could be found.

Amino Acid Sequence

Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.

The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.

Base Sequence

Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors.

Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development. Mutation of this gene (defA-1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the 'globifera' phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers. A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted 'flower specific' cDNA library. The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA-1 mutants. According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development. The 1.1 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol. wt 26.2 kd). At its N-terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c-fos) in mammals and GRM/PRTF (regulating mating type) in yeast. We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis.

Amino Acid Sequence

Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation.

The Petunia hybrida mutant RL01 is white flowering due to a genetic block in the anthocyanin pathway. The introduction of the maize A1 cDNA under the control of the CaMV 35S RNA promoter leads to the production of pelargonidin derivatives, resulting in a brick red flower phenotype. Among the transgenic petunia plants the pigmentation of the petals exhibited different expression patterns which could be categorized into the 'red', the 'variegated', and the 'white' phenotype. This system proved to be especially suitable for the investigation of gene expression by simply looking at the pigmentation pattern of the petals. The uniformity of floral pelargonidin pigmentation is inversely correlated with the number of integrated A1 copies. Furthermore, a correlation was found between the methylation status of the 35S RNA promoter and the instability of the floral pelargonidin coloration. The status of promoter methylation controlling the expression of the A1 gene seems to be influenced by the copy number and the chromosomal position of the transferred gene.

Anthocyanins

IS2-61 and IS2-611 arise by illegitimate recombination from IS2-6.

A more stable derivative of IS2-6 has been isolated, which had lost 54 bp of the 108 bp long insert characteristic of IS2-6. This new allele of IS2, IS2-61, segregates the remaining 54 bp to yield allele IS2-611. DNA sequence analysis shows that the segregation products of IS2-6 arise by recA-independent, illegitimate recombination at 9 bp long direct sequence repetitions.

Alleles

Development of a system useful for studying the formation of unstable alleles of IS2.

IS2-induced deletions of the gal control region were isolated in a plasmid carrying gal OP-308::IS2-7. This contains a 54 basepair long, unstable mini insertion within IS2, thus allowing constitutive expression of the gal structural genes. Deletion PPI is 11.9 kilobasepairs (kb) long and is Gal+ because it has retained the mini insertion. In PP4 7.2 kb DNA material including markers gal OP, chlD and pgl are deleted. PP4 has lost the mini insertion and is therefore Gal negative. DNA sequencing of the newly formed junction in PP4 reveals that the deletion terminates precisely at nucleotide 1 of IS2 and that no DNA sequence homology is involved in this IS2-mediated deletion formation. PPI segregates Gal- clones due to the loss of the mini insertion. One such segregant PPIS and PP4 both give only constitutive Gal+ revertants, which consist of the previously known mini insertions and also a new class of "supermini" inserts within IS2 of about 10 to 20 basepairs long. Therefore, PPIS and PP4 can be used to study various parameters involved in the formation of mini insertions.

Alleles

Multiple integration sites for the lactose transposon Tn 951 on plasmid RP 1 and establishment of a coordinate system for Tn 951.

Various molecules generated by transposition of the lactose transposon Tn 951 from plasmid pGC1 to plasmid RP1 were examined by DNA heteroduplex and restriction endonuclease analysis. Tn 951 was found to transpose to at least eight different sites on RP 1 in both possible orientations. A coordinate system for the lactose transposon Tn 951 is constructed.

DNA Restriction Enzymes

Integration of IS3 into IS2 generates a short sequence duplication.

The Gal+ allele IS2-43 is known to segregate Gal- clones. Among 11 Gal- segregants, one was shown to be due to the integration of IS3 into IS2-43. Precise excision of the integrated IS3 element occurred at a rate of 5 x 10(-9)/cell/generation. DNA sequence analysis revealed that the termini of the IS3 element have the relation of imperfect inverted repeats and it is now flanked by a 3bp or 4bp duplication, a size which has not been seen before with other elements.

Alleles