The histone H3 and H4 mRNAs are polyadenylated in maize

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.     

Molecular cloning of rat precursor cathepsin H and the expression of five lysosomal cathepsins in normal tissues and in a rat carcinosarcoma

1. A rat cathepsin H cDNA was isolated from a rat liver cDNA library with synthetic oligonucleotide probes. 2. DNA sequence analysis indicated that it codes for rat preprocathepsin H. 3. Using this clone together with the cDNA for cathepsins B, D, L and S as probes, the expression of five major lysosomal proteinases was investigated in ten different normal rat tissues and in a rat carcinoma. 4. The common feature of their expression is that the five cathepsins have relatively high mRNA levels in lung and kidney, suggesting that they all play important roles in organs engaged in active protein metabolism. 5. In other tissues, the concentrations of the five cathepsin mRNAs are significantly different. This may indicate that their expressions are differentially regulated and that they may have specialized functions in specific tissues. 6. The cathepsin B mRNA level is at least 2.5-fold higher in the rat W256-carcinoma than in any of the normal rat tissues surveyed. 7. In contrast, the mRNA levels for the other four cathepsins show no comparable elevations. 8. This finding is consistent with previous observations reporting a correlation between cathepsins B expression and malignant tumors.     

The two major plant plasma membrane H+-ATPases display different regulatory properties

The major plant plasma membrane H(+)-ATPases fall into two gene categories, subfamilies I and II. However, in many plant tissues, expression of the two subfamilies overlaps, thus precluding individual characterization. Yeast expression of PMA2 and PMA4, representatives of the two plasma membrane H(+)-ATPase subfamilies in Nicotiana plumbaginifolia, has previously shown that (i) the isoforms have distinct enzymatic properties and that (ii) PMA2 is regulated by phosphorylation of its penultimate residue (Thr) and binds regulatory 14-3-3 proteins, resulting in the displacement of the autoinhibitory C-terminal domain. To obtain insights into regulatory differences between the two subfamilies, we have constructed various chimeric proteins in which the 110-residue C-terminal-encoding region of PMA2 was progressively substituted by the corresponding sequence from PMA4. The PMA2 autoinhibitory domain was localized to a region between residues 851 and 915 and could not be substituted by the corresponding region of PMA4. In contrast to PMA2, PMA4 was poorly phosphorylated at its penultimate residue (Thr) and bound 14-3-3 proteins weakly. The only sequence difference around the phosphorylation site is located two residues upstream of the phosphorylated Thr. It is Ser in PMA2 (as in most members of subfamily I) and His in PMA4 (as in most members of subfamily II). Substitution of His by Ser in PMA4 resulted in an enzyme showing increased phosphorylation status, 14-13-3 binding, and ATPase activity, as well as improved yeast growth. The reverse substitution of Ser by His in PMA2 resulted in the failure of this enzyme to complement the absence of yeast H(+)-ATPases. These results show that the two plant H(+)-ATPase subfamilies differ functionally in their regulatory properties.     

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-form Trypanosoma brucei

ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)₈C(x)₅C(x)₃H zinc finger motifs. ZC3H20 is present at a low level in replicating mammalian-infective bloodstream forms, but becomes more abundant when they undergo growth arrest at high density; ZC3H21 appears only in the procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least 28 of the bound mRNAs decrease after depletion of ZC3H20, or of ZC3H20 and ZC3H21 together; their products include procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes procyclic forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form-specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.     

Early salt stress effects on the differential expression of vacuolar H(+)-ATPase genes in roots and leaves of Mesembryanthemum crystallinum.

In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H(+)-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H(+)-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.     

烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析

C2H2锌指蛋白转录因子家族在真核生物中具有重要的生物学功能,广泛参与植物叶的发生、花器官的调控、侧枝的形成及逆境胁迫等生命过程。植物C2H2锌指蛋白不仅结合DNA和RNA,而且与蛋白质之间相互作用。本研究利用普通烟草(Nicotiana tabacum)基因组数据库,运用Blastp比对,结合Pfam和SMART分析,鉴定了118条普通烟草C2H2锌指蛋白家族成员;对烟草C2H2锌指蛋白家族进行了进化树分析、结构域分析、物理化学性质分析、染色体定位、基因结构分析、三维结构分析及组织表达分析等。结果表明：不同成员的氨基酸长度差异较大;系统进化及结构域分析显示,所有C2H2家族成员可以被分为5个亚家族,同一亚家族成员之间在结构域和理化性质上呈现较高一致性;每个成员都含有C2H2结构域,在数量上存在较大差异;将所有基因家族成员定位在22条染色体上;组织表达分析表明,每个C2H2亚家族都有成员在不同组织中表达,在叶及根中有些基因的表达量较高。     

Organization of the histone H3 and H4 multigenic families in maize and in related genomes

Summary Five cloned histone H3 and H4 genes from maize have specific 5 non-transcribed regions. Blot hybridization of each 5 region to DNA from different maize inbred lines showed that the H3 and H4 multigenic families are organized into subfamilies. Each subfamily has a specific environment and contains a different (from 4–16) number of gene copies. H3 and H4 subfamilies with similar environments as those found in maize were shown to exist in the genomes of more or less related plants, including perennial teosinte, sorgho, sugar cane and Coïx. Such observations may contribute to establishing phylogenetic relationships at a molecular level between different plants and thus highlight some of the evolutionary mechanisms of the genomes of higher plants.     

Spatial Distribution of Epigenetic Modifications in Brachypodium distachyon Embryos during Seed Maturation and Germination

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in ‘matured’, ‘dry’ and ‘germinating’ embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In ‘dry’ embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.     

Regulation of genes encoding β-d-glucan glucohydrolases in barley (Hordeum vulgare)

Expression patterns of barley β - d -glucan glucohydrolase genes were monitored using cDNAs encoding isoenzymes ExoI and ExoII. The cDNAs were isolated from 5-day-old seedling libraries. The enzymes are encoded by a small gene family, in which marked differences in codon usage are evident. The cDNAs can be used as specific probes for two subfamilies of β - d -glucan glucohydrolase genes. Genes of both subfamilies are transcribed in the scutellum of germinated grain, in elongating coleoptiles, and in young roots and leaves. Low levels of mRNA for the isoenzyme ExoI gene subfamily could be detected in aleurone layers of germinated grain. Most of the β - d -glucan glucohydrolase activity can be extracted from tissues with dilute aqueous buffers. Enzyme activity is highest in young leaves and elongating coleoptiles, but is not well-correlated with mRNA levels. The expression patterns are consistent with proposed roles for β -glucan glucohydrolases in the turnover or modification of cell-wall (1→3,1→4)- β - d -glucans in elongating coleoptiles and in young vegetative tissues.     

The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.