Are there two DNA methyltransferase gene families in plant cells? A new potential methyltransferase gene isolated from an Arabidopsis thaliana genomic library.

Using the 1kb 3' terminal DNA fragment of the mouse methyltransferase cDNA as a probe and low stringent hybridisation conditions, a new potential methyltransferase (MTase) gene family was isolated from an Arabidopsis thaliana genomic DNA library. One clone (MTase-11), which gave the strongest signal at the Northern blot, was entirely sequenced (11483 bp) and further characterised. Under consideration of the likely open reading frames and our preliminary cDNA experiments we propose that the clone 11 gene encodes for an approximately 90 kD protein. As deduced form the DNA sequence this protein contains all conserved sequence motifs specific for the 5m cytosine MTases. MTase-11 gene expression was demonstrable in callus and during germination but not in one month old plants or in leaves.     

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

内皮抑素（Endostatin）是近年来新发现的一种内源性新生血管生成（Angiogenesis）抑制因子,通过抑制新血管生成而抑制肿瘤的形成和转移且不会引起耐药性,具有极高的临床应用前景。巴斯德毕赤酵母（Pichia pastoris)具有表达率高、产物可分泌、可对高等真核生物蛋白正确进行翻译后加工、遗传稳定、发酵工艺成熟等优点被用来进行重组人Endostatin的表达。本研究用PCR的方法从人胎肝cDNA文库中扩增出人Endostatin的cDNA,测序正确后转入毕赤巴斯德甲醇酵母,并获得了高效可溶型表达,用肝素亲和层析的方法进行纯化,纯化后产物经SDSPAGE薄层扫描分析纯度达987％以上,质谱测定分子量为2043kD与理论值一致,蛋白质N端序列测定结果为SPPAHTHRDFQPVLH与天然序列一致。生物活性检测证明可抑制鸡胚尿囊绒毛膜（CAM）的新生血管生成（Angiogenesis),并可抑制血管内皮细胞的增殖。因此用酵母表达系统可以得到具有生物活性的内皮抑素,经纯化后可用于进一步的生物功能和作用机理试验。     

The Arabidopsis thaliana SET-domain-containing protein ASHH1/SDG26 interacts with itself and with distinct histone lysine methyltransferases

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have central roles in cell proliferation, growth and development. In animals, PcG and trxG proteins form higher order protein complexes that contain SET domain proteins with histone methyltransferase activity, and are responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. However, whether H3K4 methyltransferase complexes exist in Arabidopsis thaliana remains unknown. Here, we make use of the yeast two-hybrid system and the bimolecular fluorescence complementation assay to provide evidence for the self-association of the Arabidopsis thaliana SET-domain-containing protein SET DOMAIN GROUP 26 (SDG26), also known as ABSENT, SMALL, OR HOMEOTIC DISCS 1 HOMOLOG 1 (ASHH1). In addition, we show that the ASHH1 protein associates with SET-domain-containing sequences from two distinct histone lysine methyltransferases, the ARABIDOPSIS HOMOLOG OF TRITHORAX-1 (ATX1) and ASHH2 proteins. Furthermore, after screening a cDNA library we found that ASHH1 interacts with two proteins from the heat shock protein 40?kDa (Hsp40/DnaJ) superfamily, thus connecting the epigenetic network with a system sensing external cues. Our findings suggest that trxG complexes in Arabidopsis thaliana could involve different sets of histone lysine methyltransferases, and that these complexes may be engaged in multiple developmental processes in Arabidopsis.     

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GAL4 regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.     

The DNMT1 target recognition domain resides in the N terminus

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122-417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.     

Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases

A cDNA encoding DNA (cytosine-5)-methyltransferase (DNA MeTase) of mouse cells has been cloned and sequenced. The nucleotide sequence contains an open reading frame sufficient to encode a polypeptide of 1573 amino acid residues, which is close to the apparent size of the largest species of DNA MeTase found in mouse cells. The carboxylterminal 570 amino acid residues of the inferred protein sequence shows striking similarities to bacterial type II DNA cytosine methyltransferases and appears to represent a catalytic methyltransferase domain. The amino-terminal portion of the molecule may be involved in regulating the activity of the carboxyl-terminal methyltransferase domain, since antibodies directed against a peptide sequence located within this region inhibits transmethylase activity in vitro. A 5200 base DNA MeTase-specific mRNA was found to be expressed in all mouse cell types tested, and cell lines known to have different genomic methylation patterns were found to contain DNA MeTase proteins of similar or identical sizes and de novo sequence specificities. The implications of these findings for an understanding of the mechanisms involved in the establishment and maintenance of methylation patterns are discussed.     

小麦中雄性不育同源序列的分离、鉴定及表达分析

利用拟南芥中已克隆的雄性核不育基因MS2和水稻中假定雄性不育蛋白的保守区域,设计一对简并引物,并在太谷核不育小麦可育株及不育株花药中进行扩增,得到了一条134bp的片段。以该片段为基础,通过电子延伸得到一个长为1604bp的序列,该序列编码的氨基酸包含一段由200个氨基酸组成的雄性不育保守区。RT-PCR结果表明,该雄性不育同源序列只在小麦可育花药中表达,而在小麦败育花药、叶片和根中不表达,说明该雄性不育同源序列为花药发育特异基因。     

Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.     

Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to theArabidopsis thaliana gene,cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with theEscherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the Ecoli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the Ecoli enzyme. In this model, the active site zinc is coordinated by His⁷³, Cys¹⁰³, Cys¹⁰⁷, and an active site hydroxyl. Additional residues that participate in catalysis, Asn⁶⁴, Glu⁶⁶, Ala⁷⁸, Glu⁷⁹, and Pro¹⁰², are conserved between the Arabidopsis and Ecoli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the Ecoli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to theA thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His₆ tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m = 226 μand 2’-deoxycytidine (K_m= 49 μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP. journal Paper Number J-18324 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 3340.