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.     

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.     

巴西橡胶树43 kD橡胶粒子膜蛋白基因的cDNA克隆及表达

对43 kD的橡胶粒子膜蛋白进行了分离纯化和其N端氨基酸序列分析,根据N端氨基酸序列,设计一简并引物,通过3'RACE(Rapid Amplification ofcDNA Ends)的方法,获得了43 kD的橡胶粒子膜蛋白的cDNA.该cDNA含有1 385个核苷酸,含有完整的阅读框架,编码381个氨基酸.在终止密码子下游,包含有一个239bp的3'非编码区.该cDNA由5个首尾相连的重复单元组成,每个单元编码76个氨基酸组成的泛素(ubiquitin)单体.编码43 kD橡胶粒子蛋白的基因具有多个拷贝,在胶乳、叶片和树皮都表达.     

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.     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。     

Alternative splicing of Pot1 (Protection of telomere)-like genes in Arabidopsis thaliana

The Pot1 (Protection of telomere 1) is a G-rich single-stranded telomeric DNA binding protein, identified first in Schizosaccharomyces pombe, and shown to play an important role in stabilizing chromosomes. Pot1-like proteins or their encoding genes have been identified from yeasts to mammals. Based on the N-terminal amino acid sequences of fission yeast and human Pot1, two Pot1-like proteins (AtPOT1-1 and AtPOT1-2) have been identified in Arabidopsis thaliana, but neither of them has been characterized yet. In this study, we amplified their full-length cDNAs by RT-PCR and found three different variants for AtPOT1-1 and two for AtPOT1-2 genes, suggesting that they are exposed to alternative splicing. Alternative splicing also occurs in human Pot1, and only one out of five splicing variants had tissue specificity. However, no tissue specificity was found for any variants of the AtPOT1-1 and AtPOT1-2 genes among buds, flowers, leaves, roots, stems, siliques and cultured cells. Northern blot hybridization indicated that AtPOT1-1 expresses more in meristematic tissues than in vegetative tissues. By western blot analysis, we found that the antibody made against the N-terminal amino acids of AtPOT1-1 recognized three different polypeptides, indicating that all three variants are being translated in Arabidopsis.