Biomedical subjects

M F Yanofsky

Publications and source records attributed to M F Yanofsky.

At least 55 records · Page 3Linked to original sources

PubMed1994

Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.

To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.

AGAMOUS Protein, Arabidopsis↗

PubMed1993

Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1.

Mutations in the AGAMOUS (AG) gene of Arabidopsis thaliana result in the conversion of reproductive organs, stamens and carpels, into perianth organs, sepals and petals. We have isolated and characterized the putative AG gene from Nicotiana tabacum, NAG1, whose deduced protein product shares 73% identical amino acid residues with the Arabidopsis AG gene product. RNA tissue in situ hybridizations show that NAG1 RNA accumulates early in tobacco flower development in the region of the floral meristem that will later give rise to stamens and carpels. Ectopic expression of NAG1 in transgenic tobacco plants results in a conversion of sepals and petals into carpels and stamens, respectively, indicating that NAG1 is sufficient to convert perianth into reproductive floral organs.

Amino Acid Sequence↗

PubMed1993

Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS.

Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral development are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG1 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAG1 encodes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAG1 outside the well-conserved MADS domain. Like AG, ZAG1 RNA accumulates early in stamen and carpel primordia. In contrast, ZAG2 expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAG1 cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG1 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts4), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.

AGAMOUS Protein, Arabidopsis↗

PubMed1992

Molecular characterization of the Arabidopsis floral homeotic gene APETALA1.

The first step in flower development is the transition of an inflorescence meristem into a floral meristem. Each floral meristem differentiates into a flower consisting of four organ types that occupy precisely defined positions within four concentric whorls. Genetic studies in Arabidopsis thaliana and Antirrhinum majus have identified early-acting genes that determine the identify of the floral meristem, and late-acting genes that determine floral organ identity. In Arabidopsis, at least two genes, APETALA1 and LEAFY, are required for the transition of an influorescence meristem into a floral meristem. We have cloned the APETALA1 gene and here we show that it encodes a putative transcription factor that contains a MADS-domain. APETALA1 RNA is uniformly expressed in young flower primordia, and later becomes localized to sepals and petals. Our results suggest that APETALA1 acts locally to specify the identity of the floral meristem, and to determine sepal and petal development.

Amino Acid Sequence↗

PubMed1992

Manipulation of flower structure in transgenic tobacco.

Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.

Amino Acid Sequence↗

PubMed1992

Vectors for plant transformation and cosmid libraries.

A series of vectors has been constructed for the purpose of introducing cloned DNAs into plant genomes, using Agrobacterium tumefaciens-mediated transformation methods. One of these vectors, pCIT20, is a plasmid that contains a multiple cloning site (MCS), and a marker (Hph) that confers hygromycin resistance to plant cells. The others are all cosmid vectors which allow insertion of up to 46 kb of plant genomic DNA, and which also contain all of the necessary sequences for A. tumefaciens-mediated plant transformation. The cosmid vectors either contain a Hph marker (pCIT30), or a kanamycin-resistance marker (pCIT101-104). Three of the cosmid vectors (pCIT30, pCIT101, and pCIT103) carry bacteriophage T7 and SP6 promoters flanking the cloning Bg/II site, for synthesis of end-specific RNAs. The end-specific RNAs may be used as probes when labeled with radioactive or biotinylated nucleotides, for example, in a chromosome-walking experiment. The other two cosmid vectors (pCIT102 and pCIT104) carry restriction sites flanking the insertion site (XhoI) for convenient release of the insert by restriction digests. These sites, in combination with sites internal to the insert, allow the generation of end fragments for subcloning or labeling probes. These vectors should be valuable for isolation and analysis of plant genes, using transformation, library screening, and chromosome-walking approaches.

Agrobacterium tumefaciens↗

PubMed1992

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.

Amino Acid Sequence↗

PubMed1992

Molecular analysis of an auxin binding protein gene located on chromosome 4 of Arabidopsis.

We have isolated a cDNA clone from Arabidopsis, At-ERabp1, for the Arabidopsis auxin binding protein located in the lumen of the endoplasmic reticulum (ER). This cDNA clone codes for a protein related to the major auxin binding protein from maize, Zm-ERabp1. A single open reading frame, 594 bases in length, predicts a protein of 198 amino acid residues and a molecular mass of 22,044 D. The primary amino acid sequence contains an N-terminal hydrophobic signal sequence of 33 amino acids. We demonstrated by in vitro studies that the At-ERabp1 protein is translocated into ER-derived microsomes. The protein was processed, and the cleavage site for the N-terminal signal peptide was determined by radiosequencing. The mature protein is composed of 165 amino acid residues, with a molecular mass of 18,641 D. The At-ERabp1 protein contains potential N-glycosylation sites (Asn46-Ile-Ser and Asn130-Ser-Thr). In vitro transport studies demonstrated cotranslational glycosylation. Retention within the lumen of the ER correlates with an additional signal located at the C terminus and represented by the amino acids Lys196-Asp-Glu-Leu, well known to be essential for active retrieval of proteins into the lumen of the ER. DNA gel blot analysis of genomic DNA revealed single hybridizing bands, suggesting that only a single At-ERabp1 gene is present in the Arabidopsis genome. Restriction fragment length polymorphism mapping indeed revealed a single locus mapping to chromosome 4.

Amino Acid Sequence↗

PubMed1991

Isolation and sequence analysis of TGA1 cDNAs encoding a tomato G protein alpha subunit.

We have isolated cDNAs for a gene coding for a G protein alpha subunit from tomato (Lycopersicon esculentum, cv. VF36). This gene, named TGA1, was isolated using a cDNA of the Arabidopsis thaliana G protein alpha subunit-encoding gene, GPA1, as a DNA probe. The sequences of four cDNA clones indicate that the deduced amino acid (aa) sequence of the gene product (TG alpha 1) has 384 aa (44906 Da). The predicted TG alpha 1 protein exhibits similarity to all known G protein alpha subunits. The aa are 84.6% identical and 93% similar (identical and conservative changes) to A. thaliana GP alpha 1, and 34% identical and 59% similar to mammalian transducins. Furthermore, it has all of the consensus regions for a GTP-binding protein. Finally, hybridizations of tomato genomic DNA indicate that TGA1 is a single-copy gene.

Amino Acid Sequence↗

PubMed1991

AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes.

The predicted products of floral homeotic genes, AGAMOUS (AG) from Arabidopsis thaliana and DEFICIENS A (DEF A) from Antirrhinum majus, have been shown previously to share strong sequence similarity with transcription factors from humans (SRF) and yeast (MCM1). The conserved sequence between these proteins is localized within a domain known to be necessary for the DNA binding and for the dimerization of SRF. We have isolated six new genes from A. thaliana, AGL1-AGL6, which also have this conserved sequence motif. On the basis of the sequence comparison between the AG and AGL genes, they can be assigned to two subfamilies of a large gene family. RNA dot blot analysis indicates that five of these genes (AGL1, AGL2, AGL4, AGL5, and AGL6) are preferentially expressed in flowers. In addition, in situ RNA hybridization experiments with AGL1 and AGL2 show that their mRNAs are detected in some floral organs but not in others. Our results suggest that these genes may act to control many steps of Arabidopsis floral morphogenesis. In contrast, the AGL3 gene is expressed in vegetative tissues as well as in flowers, suggesting that it functions in a broader range of tissues. We discuss possible roles of this gene family during the evolution of flowers.

Amino Acid Sequence↗

PubMed1990

The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors.

Mutations in the homeotic gene agamous of the plant Arabidopsis cause the transformation of the floral sex organs. Cloning and sequence analysis of agamous suggest that it encodes a protein with a high degree of sequence similarity to the DNA-binding region of transcription factors from yeast and humans and to the product of a homeotic gene from Antirrhinum. The agamous gene therefore probably encodes a transcription factor that regulates genes determining stamen and carpel development in wild-type flowers.

Amino Acid Sequence↗

PubMed1990

Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana.

We have isolated a gene coding for a G protein alpha subunit from the flowering plant Arabidopsis thaliana. This gene, named GPA1, was isolated by using a DNA probe generated by polymerase chain reaction based on protein sequences from mammalian and yeast G protein alpha subunits. The sequences of genomic and cDNA clones indicate that GPA1 has 14 exons, and the deduced amino acid sequence shows that the GPA1 gene product (GP alpha 1) has 383 amino acid residues (44,582 Da). The GP alpha 1 protein exhibits similarity to all known G protein alpha subunits--36% of its amino acids are identical and 73% are similar (identical and conservative changes) to mammalian inhibitory guanine nucleotide-binding regulatory factor alpha subunits and transducins. Furthermore, the GP alpha 1 protein has all of the consensus regions for a GTP-binding protein. The GPA1-encoded mRNA of 1.55 kilobases is most abundant in vegetative plant tissues, as determined by RNA blot analysis. Restriction fragment length polymorphism mapping experiments show that GPA1 is approximately 1.2 centimorgans from the visible marker er on chromosome 2.

Amino Acid Sequence↗

PubMed1987

Molecular characterization of the virD operon from Agrobacterium tumefaciens.

The Agrobacterium tumefaciens Ti plasmid virulence (vir) region contains at least six transcriptional units required for the efficient transfer of T-DNA to the plant genome (virA, B, C, D, E, and G). We have reported that two proteins encoded by the 5'portion of the virD operon are required for a site-specific endonuclease activity that nicks the direct repeats which flank the T-DNA. We have presented the nucleotide sequence for this portion of the operon. The nucleotide sequence of the remainder of the virD operon essential for virulence has now been determined. Two additional open reading frames encode proteins of 21.3 and 75.8 kilodaltons (kd). Translational fusions between virD2, virD3, and virD4 proteins and trpE produced fusion proteins of the size predicted from the nucleotide sequence data. We have used antisera directed against the trpE-virD2 fusion protein to detect both native virD2 protein and a virD2-lacZ fusion protein in crude extracts from Agrobacterium.

Amino Acid Sequence↗

PubMed1987

Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant.

The virulence (vir) region of Agrobacterium tumefaciens mediates the transfer of a defined segment of plasmid DNA (the T-DNA) into the plant genome. The vir genes are specifically induced by molecules produced by wounded plant cells, and virA is required for this induction. We have determined the nucleotide sequence of virA loci from limited (pTiAg162) and wide (pTiA6) host range tumor-inducing (Ti) plasmids, each of which encodes a single protein of 92,000 daltons. Using antibody directed against the virA gene product, we have localized the VirA protein to the bacterial inner membrane. VirA is homologous to at least four bacterial proteins which play a role in the transcriptional regulation of diverse families of genes. Based on its role in vir gene induction, homology to transcriptional regulators and membrane localization, we propose that VirA acts as an environmental sensor of plant-derived inducer molecules and transmits this information to the level of vir gene expression. The pTiAg162 virA locus was shown to be ineffective at directing vir gene induction, suggesting that this may in part contribute to the narrow host range conferred by this plasmid.

Bacterial Proteins↗

PubMed1987

The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA.

The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that product(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.

DNA, Bacterial↗

PubMed1987

Characterization of Agrobacterium tumefaciens strains isolated from grapevine tumors in China.

Thirteen strains of Agrobacterium tumefaciens isolated from grapevine tumors in northern China were surveyed. These strains varied in their host range properties, although all were tumorigenic on grapevines. Twelve of these strains belonged to Agrobacterium sp. biotype 3, and 11 strains resulted in the synthesis of the opine octopine in tumor tissue. Interestingly, one strain resulted in accumulation of arginine, a previously unrecognized opine, in tumor tissue. Although DNA in most of these strains showed homology to the previously characterized transferred DNA and vir loci, some virulent strains showed little or no homology to these loci. Thus, some of these strains represent widely divergent examples of Agrobacterium sp. The DNA in most strains exhibited little or no homology to a wide-host-range virA locus but did show strong homology to a limited-host-range virA locus. This finding further supports the idea that Agrobacterium strains associated with grapevines may have a specific virA locus.

China↗

PubMed1987

Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.

DNA, Bacterial↗

PubMed1987

Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA.

Transfer and integration of a defined region (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is essential for tumor formation. We used a physical assay to study structural changes induced in Agrobacterium T-DNA by cocultivation with plant cells. We show that nicks are introduced at unique, identical locations in each of the 24-base-pair imperfect direct repeats which flank the T-DNA and present evidence that a linear, single-stranded molecule is generated. We propose that these changes result from processing of the T-DNA for transfer and that they occur by a mechanism similar to DNA processing during conjugative DNA transfer between bacteria.

Cell Line↗