Molecular analysis of the NAC gene family in rice

Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development.     

金鱼藤中ApNAP基因的克隆和表达模式分析

金鱼藤(Asarina procumbens)是玄参科金鱼藤属多年生藤本植物,在园林中常用于垂直绿化,是良好的观赏植物,但金鱼藤叶片衰老后很快干枯并脱落,仅留光秃的茎蔓,使其观赏价值大大降低。AtNAP是拟南芥NAC基因家族中与衰老相关的转录因子,在调控叶片衰老过程中有重要的作用。本研究以金鱼藤衰老叶片为材料,运用RT-PCR和RACE技术克隆金鱼藤中ApNAP基因cDNA序列。结果显示,该基因位于细胞核中,cDNA全长为1341 bp,开放阅读框为936 bp,编码311个氨基酸,相对分子量为35.21 kD,等电点为9.13;ApNAP基因组全长1526 bp,含有2个内含子和3个外显子。多重序列比对表明,ApNAP属于NAC转录因子家族。系统进化树分析显示,ApNAP属于NAP亚组,与AtNAM的同源关系最近。实时荧光定量PCR结果表明,受自然状态下衰老和黑暗处理的诱导,ApNAP基因表达量增加,这进一步说明ApNAP基因参与调控金鱼藤叶片衰老的过程。该基因的分离对利用转基因技术延缓金鱼藤叶片衰老,提高金鱼藤在园林绿化中的观赏价值具有实际意义。     

The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins

In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown. CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins. This study investigated the molecular basis for their activities. Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus. Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity. Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located. Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes.     

Genomic structure of keratinocyte transglutaminase. Recruitment of new exon for modified function.

The gene for keratinocyte transglutaminase (TGK) spans 14 kilobase pairs and contains 15 exons. Many features of the TGK gene are very similar, if not identical, to those of the gene encoding the catalytic subunit of human clotting factor XIII: they have the same number of exons, corresponding introns always interrupt the coding region in the same phase of the codon, and most exons are of similar size (10 or 15 are exactly the same size). In these respects, the TGK and factor XIII catalytic subunit genes resemble each other more than either resembles the gene for erythrocyte band 4.2, a noncatalytic transglutaminase superfamily member. Exon II in both the TGK and factor XIII genes encodes an amino-terminal extension of nonhomologous sequence which in each protein confers a specialized function (membrane anchorage or activation of cross-linking, respectively). This suggests that the evolution of these genes included recruitment of a new exon to modify the enzyme action. Southern blots of genomic DNA reveal the presence of a TGK-like gene in birds, amphibians, and fish, but not in flies.     

Auxin response factor family in rice

We isolated 11 rice genes homologous to the genes encoding auxin response factors (ARFs) in Arabidopsis. All of the genes encoded a well-conserved amino acid sequence in the N-terminal region, which is considered to be a DNA-binding domain (DBD). Phylogenetic analysis based on comparison of the DBDs indicated that rice has one or two closely related orthologs corresponding to a given respective ARF gene in Arabidopsis. We also analyzed the amino acid sequences of another conserved domain in the C-terminal conserved domain (CTD), which was shared by almost all the rice ARFs, with the exception of OsETTIN1 and OsETTIN2. These results agreed well with the evolutionary relationship deduced from the DBD comparison. In contrast to many ARFs, OsETTIN1 and OsETTIN2 do not contain the conserved C-terminal domain, but do share another consensus motif that is also found in Arabidopsis ETTIN. All of the above observations indicate that rice has functionally diversified ARF genes whose structures and functions correspond to those of various Arabidopsis ARFs, with one or two rice ARFs corresponding to a given Arabidopsis ARF. Thus, auxin signal transduction mechanisms may be well conserved between monocot and dicot plants.     

棉花生长素应答因子克隆和序列分析

通过电子克隆和RACE相结合的方法,从陆地棉中克隆到一个新ARF基因。序列分析表明,该基因序列全长为2393其中包括87bp的5′非编码区(5′UTR),1941bp的蛋白质编码区,终止密码子TAA和362bp的3′非编码区。该基因可编码647个氨基酸的蛋白质,分子量为71.9kD,等电点(PI)为8.2。该基因含有一个与拟南芥中ARF基因相似的B3结构域和一个Auxin_resp结合位点,表明该基因与拟南芥ARF基因有很高的同源性,推测具有相似或相同的功能。     

Structural isoforms of a membrane transport protein from Leishmania enriettii.

A membrane transport protein of the glucose transporter superfamily from Leishmania enriettii is encoded by a family of tandemly repeated genes. The first gene in this tandem repeat codes for a structural isoform that contains a unique amino-terminal hydrophilic domain, probably located in the cytoplasm; the remainder of the protein is identical to the polypeptide encoded by the internal genes in the tandem repeat. The unique isoform is represented by a distinct stable RNA.     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

The cellular retinol binding protein II gene. Sequence analysis of the rat gene, chromosomal localization in mice and humans, and documentation of its close linkage to the cellular retinol binding protein gene

Cellular retinol binding protein II (CRBP II) is an abundant, 134-residue protein present in the small intestinal epithelium. It is thought to participate in the uptake and/or intracellular metabolism of vitamin A. We have isolated and sequenced the rat CRBP II gene. Its four exons span 0.65 kilobases and are interrupted by three introns with an aggregate length of 19.5 kilobases. Southern blot hybridization analysis indicated that this gene is highly conserved in rats, mice, and humans. CRBP II belongs to a protein family that contains eight known members. Computer-assisted comparative sequence analyses indicated that a region of internal homology spans its first two exons and that oligopeptide domains specified by these first two exons exhibit significant homology to all other family members as well as to a portion of the all-trans-retinol binding domain that has previously been defined in serum retinol binding protein. The CRBP II gene was mapped in mice using recombinant inbred strains and restriction fragment length polymorphisms. It is located on chromosome 9 within 5.3 centimorgans of the phosphoglucomutase-3 locus and is closely linked (within 3.0 centimorgans) to the gene specifying a highly homologous intracellular retinol binding protein known as CRBP. Mouse-human somatic cell hybrids were used to determine that both the CRBP and CRBP II genes are located on human chromosome 3.     

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine

The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.