Structure and Evolution of Genes Encoding Polyubiquitin and Ubiquitin-like Proteins in Arabidopsis Thaliana Ecotype Columbia

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, K(s) value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.     

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 -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.     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.     

Expression of. Arabidopsis tryptophan biosynthetic pathway genes: effect of the 5' coding region of phosphoribosylanthranilate isomerase gene

There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAI1 or PAI3 was 60—100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference). The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by t     

A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems of transgenic Arabidopsis

The tissue-specific pattern of expression directed by the H4A748 Arabidopsis histone promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing H4A748-GUS gene fusions. As determined by fluorimetric and histochemical tests, the H4A748 promoter directs preferential expression in meristems of young seedlings and adult plants. The low activity found in nonproliferating tissues may relate to basal constitutive expression of the histone promoter and/or to endoreduplication occurring in some tissues. The endogenous histone mRNA levels parallel the GUS activity found in different tissues. Analysis of the regulatory properties of 5' deleted promoters showed that multiple positive elements exist between -900 and -219 and that the proximal region of the promoter to -219 is sufficient to establish the full tissue-specific pattern of expression. Further deletion to -93 nearly abolished the promoter activity thus suggesting that the 126 bp fragment located between -219 and -93 contains the elements responsible for the specific expression pattern. The presence of several remarkable sequences within this fragment is discussed.     

T-DNA tagging and characterization of a cryptic root-specific promoter in Arabidopsis

From a T-DNA tagged Arabidopsis population, a line, M-57 showing GUS (beta-glucuronidase) expression in the vascular regions of young roots was identified. Southern analysis revealed presence of a single T-DNA insert. Using inverse PCR, the plant sequence flanking the T-DNA insertion was cloned. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l-Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. A 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies. This cryptic promoter element does not show the presence of any known root-specific promoter element.