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L Comai

Publications and source records attributed to L Comai.

At least 37 records · Page 2Linked to original sources
PubMed1998

Agrobacterium VirD2 protein interacts with plant host cyclophilins.

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

Bacterial Proteins↗
PubMed1998

A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis.

Chromodomains are thought to mediate protein-protein interactions between chromatin components. We have detected a chromodomain embedded within the catalytic region of a predicted Arabidopsis DNA methyltransferase that is diverged from other eukaryotic enzymes. The 791 residue "chromomethylase" (CMT1) is encoded by a floral transcript that is spliced from 20 exons and is present at only approximately 1/10(-7) of total mRNA. Genomic sequencing reveals an ancient haplotype split at CMT1 between Col-0 + Metz and the other ecotypes examined. In the Col-0 + Metz haplotype, alternative mRNA processing at intron 13 truncates the coding region. In Ler, RLD, and No-0, similar truncation is caused by insertion of an intact retrotransposon, Evelknievel, which is present as a single copy in Ler and RLD and is currently methylated and inactive. Evelknievel is found at this site on a single branch that connects the Ler, RLD, and No-0 ecotypes but is absent from the genomes of all other ecotypes examined. A stop codon within exon 6 of the Metz ecotype confirms that CMT1 is nonessential. Nevertheless, comparison to CMT1 of Cardaminopsis arenosa, an outcrossing relative, indicates conservation for DNA methyltransferase function. We discuss how allelic diversity of CMT1 may reflect loosened selective constraints in a self-fertilizing species such as Arabidopsis thaliana.

Amino Acid Sequence↗
PubMed1997

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA.

A cellular protein, previously described as p35/38, binds to the complementary (-)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(-)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

Animals↗
PubMed1997

SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription.

SV40 large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle and can stimulate cell proliferation. To accomplish this, large T antigen has to control the expression of cellular genes involved in cell cycle progression and cell growth. rRNA synthesis by RNA polymerase I (Pol I) is tightly associated with cell growth and proliferation, and previous studies indicated that large T antigen up-regulates RNA Pol I transcription in SV40-infected cells. How this process occurs is currently unclear. To investigate the mechanisms of large T antigen stimulation of RNA Pol I transcription, we have established an in vitro transcription system that is responsive to large T antigen. Here, we show that recombinant large T antigen stimulates Pol I transcription reconstituted with purified RNA Pol I, UBF, and the TBP/TAF complex SL1. Immunoprecipitation experiments revealed that large T antigen directly binds to SL1, in vitro, as well as in SV40-infected cells. In addition, our data indicate that this interaction occurs by direct association with three SL1 subunits, namely TBP, TAF(I)48, and TAF(I)110. Transcription studies with large T antigen deletion mutants show that the 538-amino-acid amino-terminal domain is necessary for full stimulation of Pol I transcription. Importantly, mutants that no longer bind to SL1 are also unable to stimulate Pol I transcription. This indicates that recruitment of large T antigen to the rRNA promoter by SL1 constitutes a crucial step in the activation process. Taken together with recent studies on large T antigen activation of RNA Pol II transcription, these results suggest that viral modulation of genes involved in cell proliferation involves direct targeting of promoter-specific TBP/TAF complexes (i.e., SL1 or TFIID) by large T antigen.

Antigens, Polyomavirus Transforming↗
PubMed1996

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions.

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs, ARS1 and CENIII, showed that the HSC80 5'MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5'MAR can outcompete the 3'MAR and not vice versa. Last, we observed that the interaction of the 3'MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.

Binding, Competitive↗
PubMed1996

Variegation and silencing of the Heat Shock Cognate 80 gene are relieved by a bipartite downstream regulatory element.

Transgenes are important tools for plant molecular biologists. However, some aspects of their regulation are not completely understood. First, the contribution of elements separate from the promoter has been documented for several genes, but a general requirement for such elements is still unclear. Second, transgene expression can be subject to variability from chromosomal position effects and from epigenetic effects, often due to the presence of repeated sequences. While studying the regulation of the developmentally expressed Heat Shock Cognate 80 (HSC80) gene, several peculiarities were found which involve both of these questions. First, HSC80 transgenes are subject to silencing and variegation at a high frequency. Second, a bipartite element downstream of the transcriptional start is necessary to achieve efficient expression from the HSC80 promoter in stable transformants. Finally, this bipartite element is not required for expression in a transient system, where the promoter alone is sufficient. These results together support a role for chromatin structure in the regulation of HSC80.

Arabidopsis↗
PubMed1996

The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs.

In cycling cells, the rDNAs are expressed from telophase to the end of G2 phase. The early resumption of rDNA transcription at telophase raises the question of the fate of the rDNA transcription machinery during mitosis. At the beginning of mitosis, rDNA transcription is arrested, and the rDNAs are clustered in specific chromosomal sites, the nucleolar organizer regions (NOR). In human cells, we demonstrate that the rDNA transcription machinery, as defined in vitro, is colocalized in some NORs and absent from others whatever the mitotic phase: RNA polymerase I and the RNA polymerase I transcription factors, upstream binding factor and promoter selectivity factor (as verified for TATA-binding protein and TATA-binding protein-associated factor for RNA polymerase I [110]), were colocalized in the same NORs. The RNA polymerase I complex was localized using two different antibodies recognizing the two largest subunits or only the third largest subunit, respectively. These two antibodies immunoprecipitated the RNA polymerase I complex in interphase cells as well as in mitotic cells. These results clearly indicated that the RNA polymerase I complex remained assembled during mitosis. In addition, RNA polymerase I and the transcription factors varied in the same proportions in the positive NORs, suggesting stoichiometric association of these components. The fact that the rDNA transcription machinery is not equally distributed among NORs most likely reflects the implication of the different NORs during the subsequent interphase. Indeed, we demonstrate that only positive NORs exhibit transcription activity at telophase and that the level of transcription activity is related to the amount of rDNA transcription machinery present in the NOR. We propose that assembly of rDNA transcription machinery preceding mitosis determines expression of the rDNAs at the beginning of the next cell cycle. Consequently, the association of rDNAs with the rDNA transcription machinery defines the "active" NORs and the level of activity at the transition telophase/interphase.

Cell Nucleolus↗
PubMed1995

The REVOLUTA gene is necessary for apical meristem development and for limiting cell divisions in the leaves and stems of Arabidopsis thaliana.

The form of seed plants is determined by the growth of a number of meristems including apical meristems, leaf meristems and cambium layers. We investigated five recessive mutant alleles of a gene REVOLUTA that is required to promote the growth of apical meristems and to limit cell division in leaves and stems of Arabidopsis thaliana. REVOLUTA maps to the bottom of the fifth chromosome. Apical meristems of both paraclades (axillary shoots) and flowers of revoluta mutants frequently fail to complete normal development and form incomplete or abortive structures. The primary shoot apical meristem sometimes also arrests development early. Leaves, stems and floral organs, in contrast, grow abnormally large. We show that in the leaf epidermis this extra growth is due to extra cell divisions in the leaf basal meristem. The extent of leaf growth is negatively correlated with the development of a paraclade in the leaf axil. The thickened stems contain extra cell layers, arranged in rings, indicating that they may result from a cambium-like meristem. These results suggest that the REVOLUTA gene has a role in regulating the relative growth of apical and non-apical meristems in Arabidopsis.

Alleles↗
PubMed1994

Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits.

RNA polymerase I and II transcription factors SL1 and TFIID, respectively, are composed of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) responsible for promoter recognition. How the universal transcription factor TBP becomes committed to a TFIID or SL1 complex has not been known. Complementary DNAs encoding each of the three TAFIs that are integral components of SL1 have not been isolated. Analysis of subunit interactions indicated that the three TAFIs can bind individually and specifically to TBP. In addition, these TAFIs interact with each other to form a stable TBP-TAF complex. When TBP was bound first by either TAFI110, 63, or 48, subunits of TFIID such as TAFII250 and 150 did not bind TBP. Conversely, if TBP first formed a complex with TAFII250 or 150, the subunits of SL1 did not bind TBP. These results suggest that a mutually exclusive binding specificity for TBP intrinsic to SL1 and TFIID subunits directs the formation of promoter- and RNA polymerase-selective TBP-TAF complexes.

Amino Acid Sequence↗
PubMed1994

Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits.

Initiation of ribosomal RNA synthesis by RNA polymerase I requires the promoter selectivity factor SL1, which consists of the TATA-binding protein, TBP, and three associated factors, TAFIS 110, 63, and 48. Here the in vivo and in vitro assembly of functional SL1 complexes from recombinant TAFIS and TBP are reported. Complexes containing TBP and all three TAFIS were as active in supporting transcription from the human ribosomal RNA gene promoter as endogenous SL1, whereas partial complexes without TBP did not efficiently direct transcription in vitro. These results suggest that TAFIS 110, 63, and 48, together with TBP, are necessary and sufficient to reconstitute a transcriptionally active SL1 complex.

DNA-Binding Proteins↗
PubMed1992

The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1.

We have previously shown that the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) are required for regulated transcriptional initiation by RNA polymerase II. Here we report the biochemical properties of the RNA polymerase I promoter selectivity factor, SL1, and its relationship to TBP. Column chromatography and glycerol gradient sedimentation indicate that a subpopulation of TBP copurifies with SL1 activity. Antibodies directed against TBP efficiently deplete SL1 transcriptional activity, which can be restored with the SL1 fraction but not purified TBP. Thus, TBP is necessary but not sufficient to complement SL1 activity. Analysis of purified SL1 reveals a complex containing TBP and three distinct TAFs. Purified TAFs reconstituted with recombinant TBP complement SL1 activity, and this demonstrates that TBP plus novel associated factors are integral components of SL1. These findings suggest that TBP may be a universal transcription factor and that the TBP-TAF arrangement provides a unifying mechanism for promoter recognition in animal cells.

Antibodies↗
PubMed1992

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A.

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.

2S Albumins, Plant↗
PubMed1992

Developmental expression of tomato heat-shock cognate protein 80.

Heat-shock protein 80 (HSP80) is a major heat-shock protein induced in yeast and animals both by heat shock and by specific developmental events. In plants, a heat-shock-induced HSP80 cDNA has been described, although no information concerning developmental regulation of HSP80 genes is available. We have characterized a tomato (Lycopersicon esculentum) gene encoding a typical HSP80 protein. This gene, called HSC80, is interrupted by two introns, 995 and 109 bp long. Northern blot analyses and in situ RNA hybridization show that HSC80 mRNA is abundant in shoot and root apices and in fertilized ovaries up to 6 d postanthesis but is rare in mature leaves. Heat shock increased mRNA levels in mature leaves but only 3-fold. Developmental regulation of the HSC80 gene was confirmed by fusing 2 kb of its 5' region to the beta-glucuronidase reporter gene and introducing the chimeric gene into tomatoes. The roots of transformants showed high beta-glucuronidase expression in the apex and in lateral root primordia but not in mature tissue. Expression in the shoot was up to 10-fold higher in the apex than in mature leaves. Thus, HSC80 is preferentially expressed in shoot and root apices during normal development.

Journal Article↗
PubMed1992

Expression of a Brassica napus Malate Synthase Gene in Transgenic Tomato Plants during the Transition from Late Embryogeny to Germination.

To study gene regulation during the transition from late embryogeny to germination, we have analyzed the expression of a gene encoding the glyoxylate cycle enzyme malate synthase in transgenic tomato (Lycopersicon esculentum) plants. We have shown that although there are at least four classes of malate synthase genes in Brassica napus L., one gene is expressed at a high level during both late embryogeny and postgermination. Analyses of transgenic tomato plants containing the expressed B. napus gene along with 4.7 and 1.0 kilobase pairs of 5' and 3' flanking sequences, respectively, confirmed that a single gene is expressed at both stages of development. Furthermore, localization studies have shown that mRNA encoded by the B. napus gene is distributed throughout the tissues of a mature embryo but is not detected in the vascular cylinder of a seedling. We conclude that the sequences required to qualitatively regulate the gene correctly over the plant life cycle are present within the transferred gene and/or flanking regions. Moreover, the malate synthase gene is regulated differently during late embryogeny and postgermination in the developing vascular cylinder.

Journal Article↗