Characterization of pollen polygalacturonase encoded by several cDNA clones in maize

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes form a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.     

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.     

通过cDNARDA法分离和识别盐藻(Dunaliella salina)盐胁迫相关基因

采用cDNA代表性差异分析 (RDA)技术 ,对盐藻在盐胁迫时差异表达的基因进行了分离鉴定 .在分离到的 10个基因中 ,有 5个与已知基因同源 (包括叶绿素a b结合蛋白基因、蛋白磷酸酶I催化亚基基因和 3个核糖体蛋白基因 ) ,还有 5个未知功能基因则是首次在盐藻中被分离 .值得注意的是 ,所有这 5个已知基因的功能都与细胞分裂或盐胁迫有关 .结果表明 :取样时盐藻细胞仍处于恢复阶段 ,所分离到的基因对于盐藻耐盐可能具有重要意义 ;蛋白磷酸酶I的下调表达可能是盐藻调节离子平衡的一个重要过程和细胞分裂受阻的原因所在 ;盐藻减缓细胞分裂速度可能是为了减少能量消耗 ,以留出足够的能量来应对盐胁迫 ;其它 5个未知基因可能也与盐藻适应盐胁迫机制有关 .     

Isolation and characterization of cDNA clones for mouse Ly-9.

We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.     

Isolation of pathogen/stress-inducible cDNAs from alfalfa by mRNA differential display

Differential display of mRNA was used to isolate a full-length (SRG1) and a partial (SRG2) alfalfa cDNA induced during infection with the fungal pathogen Colletotrichum trifolii. The deduced amino acid sequences are similar to each other and resemble plant defense-related proteins and tree pollen allergens. SRG1 is a member of a gene family in alfalfa, which may also include the putative defense-related gene PR10. Unlike many defense-related genes described in similar systems, expression of SRG1-like genes does not correlate with resistance to C. trifolii. We speculate SRG1 is induced in response to plant stress.     

喉癌差异表达cDNA序列的分离与初步鉴定

分离和克隆人喉癌中新的相关基因将有助于提示喉癌的易感性与癌变机制。运用ｍＲＮＡ差异显示法对２例成人喉癌组织及配对癌旁正常组织的基因表达进行研究,分离到３５个差异显示片段;用反向Ｎｏｒｔｈｅｒｎ点杂交筛选到６个差异片段,经克隆、测序和匹配分析,得１２条不同ｃＤＮＡ序列,其中４条为新基因序列,另外８条与已知基因高度同源。将１２条ｃＤＮＡ序列固定在膜上,用来自喉癌和配对正常组织的总ｃＤＮＡ探针与其杂交和     

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a “hybrid” gene is able to direct the synthesis of a 25-kDal “hybrid” protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Isolation and characterization of cDNA clones for human skeletal muscle alpha actin.

Two cDNA libraries corresponding to polyA+ RNA from human adult skeletal muscle have been constructed by cloning in the PstI site of pBR322. Skeletal alpha actin cDNA clones have been isolated and characterized. Three of these plasmids have overlapping inserts which together contain the complete 5' non-coding and protein-coding region and part of the 3' untranslated region. Determination of the sequence of the cloned cDNA confirms the complete conservation in human of the amino-acid sequence of skeletal alpha actin compared to the rabbit or rat proteins. The 5' untranslated region, but not the 3' untranslated region, shows good homology with the corresponding one in the rat gene. Analysis of changes at silent sites within the protein-coding region suggests that the divergence of skeletal and cardiac alpha actin took place much earlier than the mammalian radiation. The plasmids described here have been used as probes to detect the homologous gene among the about thirty actin sequences present in the human genome.     

Isolation and characterization of Metolachlor-resistant mutants of Saccharomyces cerevisiae

The herbicide Metolachlor (-chloroacetamide group) inhibits the growth of Saccharomyces cerevisiae on complete, minimal, and non-fermentative media. Spontaneous and induced resistant mutants showed monogenic segregation patterns. Among the resistant clones, 70% were recessives, 16.4% were partially dominants and 13.4% were dominants. The spontaneous partially dominant mutation Mtc1 was mapped on linkage group XV at 33.3cM from ade2 and 31.7cM from his3, in a region that is characterized by the presence of several resistant genes. The recessive mutation mtc2 was located on chromosome IV. Although all the mutants had the ability to grow in the presence of the herbicide, they remained affected in their respiration efficiency, indicating two different mechanism of action of Metholachor on yeast cells.