The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.     

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.     

A role for the divergent actin gene, ACT2, in nuclear pore structure and function

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.     

Intron loss and gain during evolution of the catalase gene family in angiosperms

Angiosperms (flowering plants), including both monocots and dicots, contain small catalase gene families. In the dicot, Arabidopsis thaliana, two catalase (CAT) genes, CAT1 and CAT3, are tightly linked on chromosome 1 and a third, CAT2, which is more similar to CAT1 than to CAT3, is unlinked on chromosome 4. Comparison of positions and numbers of introns among 13 angiosperm catalase genomic sequences indicates that intron positions are conserved, and suggests that an ancestral catalase gene common to monocots and dicots contained seven introns. Arabidopsis CAT2 has seven introns; both CAT1 and CAT3 have six introns in positions conserved with CAT2, but each has lost a different intron. We suggest the following sequence of events during the evolution of the Arabidopsis catalase gene family. An initial duplication of an ancestral catalase gene gave rise to CAT3 and CAT1. CAT1 then served as the template for a second duplication, yielding CAT2. Intron losses from CAT1 and CAT3 followed these duplications. One subclade of monocot catalases has lost all but the 5'-most and 3'-most introns, which is consistent with a mechanism of intron loss by replacement of an ancestral intron-containing gene with a reverse-transcribed DNA copy of a fully spliced mRNA. Following this event of concerted intron loss, the Oryza sativa (rice, a monocot) CAT1 lineage acquired an intron in a novel position, consistent with a mechanism of intron gain at proto-splice sites.     

Paramecium tetraurelia encodes unconventional actin containing short introns

The polymerase chain reaction was used to amplify and clone an actin gene fragment from Paramecium tetraurelia. This DNA fragment was 1,138 bp long, more than 96% of the actin coding sequence, and contained four in-frame UAA codons and two small introns located at positions unique in the actin intron catalogue. This is the first report for the phylum Ciliophora of an actin gene containing introns. The deduced amino acid sequence of this actin fragment shared 58-77% identity with other actins. When compared with rabbit alpha-muscle actin, similarities were observed mainly in subdomains 1 and 3, whereas subdomains 2 and 4 appeared to be more divergent.     

The Arabidopsis 14-3-3 multigene family

The 14-3-3 proteins are ubiquitous eukaryotic proteins and are encoded by a gene family in many species. We examined the 14-3-3 gene family in Arabidopsis thaliana and found that it contains 10 members. Four new cDNAs, GF14 epsilon, GF14 kappa, GF14 mu, and GF14 nu, and two new genomic clones of GF14 phi and GF14 nu were isolated and characterized. Together with the six previously described 14-3-3 isoforms in Arabidopsis, they constitute a complete family of 10 distinct 14-3-3 proteins of 248 to 268 amino acids. Phylogenetic analysis revealed the presence of two ancient, distinct 14-3-3 gene classes in Arabidopsis and other plants. The epsilon forms diverged early from the other plant isoforms, and plant 14-3-3 genes displayed a different evolutionary course from that of mammals.     

Interactions of lactone, carboxylate and self-aggregated forms of camptothecin with human and bovine serum albumins

Highly degenerate primers to conserved regions of the eukaryotic phosphoinositol-specific phospholipase C (PLC) were used to amplify fragments of plant PLCs from Arabidopsis thaliana genomic DNA. Eight completely different fragment sequences that showed high homology to PLCs of both animals and plants were isolated. The variation between these putative PLCs was high and suggests that, like animals, plants have multiple isoforms of PLC. Using one of the PCR clones, we isolated a corresponding full-length Arabidopsis PLC gene (ATHATPLC1G), and sequence analysis indicated that it was most like a delta-type PLC. This gene is 2.5 kb and contains seven introns, all but one of which has intron/exon border sequences that conform to the Arabidopsis consensus. The structural complexity of the gene is relatively simple compared to mammalian beta-type PLCs that can be 15 kb long with up to 30 introns. The plant gene is a single copy and was mapped to four Arabidopsis YACs, one located on chromosome 2. The promoter region contained two TATA-like elements at -43 and -185 and other putative regulatory elements that suggest that this PLC is hormonally regulated. This is the first plant PLC gene and the first delta type-PLC gene from a higher organism to be sequenced.     

Open questions in photobiology. IV. Photoaging of the skin

Two isoforms of troponin C (TnC) are encoded by distinct single copy genes. Expression of fast TnC is restricted to the skeletal muscle, whereas the slow isoform is expressed in both skeletal and cardiac muscle. Chicken slow TnC (cTnC) gene is also expressed in some non-muscle tissues like the liver and the brain. Expression of cTnC gene is regulated by two distinct enhancers in cardiac and skeletal muscles. The cardiac specific enhancer is located in the immediate 5' flanking region (bp-124 to -79) of the murine cTnC gene whereas the skeletal enhancer is located within the first intron (bp 997 to 1141). In the present study we have examined how cTnC gene expression is regulated in the chicken liver. Transient transfection of liver cells with CTnC-CAT reporter constructs containing various regions of the murine cTnC gene showed that its expression in chicken liver is regulated by the cardiac specific enhancer. Furthermore, electrophoretic mobility shift assays using synthetic oligonucleotides corresponding to both CEF-1 and CEF-2 regions of the murine cardiac enhancer revealed formation of specific DNA-protein complexes. Ultraviolet light induced covalent linking of nuclear proteins to CEF-1 and CEF-2 oligomers were used to examine the nature of the cardiac enhancer binding polypeptides; one polypeptide of 48 kDa appeared to bind to both CEF-1 and CEF-2 sequences.     

An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea

A genomic clone of RbcS was isolated from an alfalfa (Medicago sativa L. cv. Apica) genomic library and characterized. Although this clone has structural features similar to a functional gene, the second exon is interrupted by a stop codon and thus is not fully translatable in the plant. Sequence analysis of the 5' and 3' noncoding regions of RbcSK-1A showed a high sequence homology to the flanking sequences of the RbcS-3A gene from pea. The regions of homology contain many important cis-regulatory elements shown to be essential for regulation of the RbcS-3A gene in pea. The promoter of this alfalfa rubisco clone was used in a translational fusion to test its ability to control the expression of the GUS reporter gene in an homologous nuclear background. High levels of GUS enzyme activity were recorded. These strong levels are comparable to some exceptionally high levels produced in other studies following the use of photosynthesis gene promoters in fusions with the GUS reporter gene.     

Members of the Arabidopsis actin gene family are widely dispersed in the genome

Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number. The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes. Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation. All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes. The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5). Even the members of three closely related and recently duplicated pairs of actin genes are unlinked. Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evolutionary history. Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.     

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with approximately 81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.