Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)⁺ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening.

A membrane-associated lipoxygenase from breaker-stage fruit of tomato (Lycopersicon esculentum Mill.) was purified and partially sequenced. Using degenerate oligonucleotides corresponding to portions of this sequence, a cDNA was amplified by PCR and used to screen a breaker fruit cDNA library. Two clones, tomloxA and tomloxB, were isolated and one of these (tomloxA) corresponded to the isolated protein. Genomic clones were isolated and sequence data from these were used to obtain the 5' ends of the cDNAs. The 2.8-kb cDNAs encode proteins that are similar in size and sequence to each other and to other plant lipoxygenases. DNA blot analysis indicated that tomato contains three or more genes that encode lipoxygenase. RNA blot analysis showed that tomloxA is expressed in germinating seeds as well as in ripening fruit, where it reached its peak during breaker stage. tomloxB appears to be fruit specific and is at its highest level in ripe fruit.     

Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins

The nucleotide sequence and derived amino acid sequence were determined for a full-length version of the tomato cDNA clone, pTOM75, the mRNA for which has previously been shown to accumulate in roots, ripening fruit and senescing leaves. Computer analysis of the predicted protein product, which we have named tomato ripening-associated membrane protein (TRAMP) indicates strong homology to known transmembrane channel proteins from other organisms. Northern analysis showed that this gene was induced by waterstress and that this induction was unaffected in an ABA-deficient genetic back-ground.     

Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger

Roots of several solanaceous plants produce anticholinergic alkaloids, hyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11), catalyzes hydroxylation of hyoscyamine in the biosynthetic pathway leading to scopolamine. We report here on the isolation of cDNA clones encoding the hydroxylase from a cDNA library made from mRNA of the cultured roots of Hyoscyamus niger. The library was screened with three synthetic oligonucleotides that encode amino acid sequences of internal peptide fragments of the purified hydroxylase. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame that encodes 344 amino acids (Mr = 38,999). All 12 internal peptide fragments determined in the purified enzyme were found in the amino acid sequence deduced from the cDNA. With computer-aided comparison to other proteins we found that the hydroxylase is homologous to two synthases involved in the biosynthesis of beta-lactam antibiotics in some microorganisms and the gene products of tomato pTOM13 cDNA and maize A2 locus which had been proposed to catalyze oxidative reactions in the biosynthesis of ethylene and anthocyan, respectively. RNA blotting hybridization showed that mRNA of the hydroxylase is abundant in cultured roots and present in plant roots, but absent in leaves, stems, and cultured cells of H. niger.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Isolation of cDNA clones of rabbit angiotensin converting enzyme: identification of two distinct mRNAs for the pulmonary and the testicular isozymes

We have isolated cDNA clones of rabbit angiotensin converting enzyme. These clones were isolated by antibody-screening of a lambda gt11 expression library made from rabbit testicular mRNA. The 2.6 kb insert of one such clone was subcloned in pBR322 and used as a hybridization probe. Out of the twenty independently isolated clones only seven hybridized with this probe suggesting that these clones belong to at least two families. Northern analysis revealed the presence of a 2.6 kb mRNA in rabbit testes and a 5.0 kb mRNA in rabbit lungs which hybridized strongly with this probe. These results indicate that the two tissue-specific isozymic forms of angiotensin converting enzyme are encoded by two distinct mRNAs which share sequence homologies.     

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue

Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage M13 mp8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of RNA complementary to two of the clones show a marked increase during the 24h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated RNA per microgram of total RNA which increases 2.5-fold during the same period. The would-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants.Abbreviations cDNA complementary DNA - poly(A) polyadenosine - poly(A)⁺ RNA polyadenylated RNA - poly(U) polyuridine     

Abundance patterns of lily pollen cDNAs: characterization of three pollen-preferential cDNA clones

Twenty-five clones were randomly selected from a mature pollen cDNA library of Easter lily (Lilium longiflorum Thunb.) in order to study the abundance of pollen-expressed mRNAs and the functional roles of the proteins encoded by these mRNAs. Plaque hybridization experiments were conducted to estimate indirectly the expression level of the mRNAs. Based on the hybridization frequency in the mature pollen library, the cDNA clones were divided into three abundance groups. Eight clones belonged to a high abundance class in which each cDNA clone was present in the mature lily pollen library at a frequency between 0.3 and 3%. Six of these clones were not found in cDNA libraries made from carpel, leaf, or root, suggesting that they are preferentially expressed in pollen. Fourteen clones belonged to a medium abundance class and were present in the mature pollen library at a frequency between 0.01 and 0.08%. The remaining three clones, which were present at a frequency below 0.01%, were grouped as a low abundance class. Almost all of the cDNA clones which belong to either the medium or low abundance class were also detected in the leaf library. Northern blot hybridization with three of the highly abundant cDNA clones confirmed their preferential expression in anther. In situ hybridization experiment with one of the clones showed the pollen-specific expression of the clone in mature anther. DNA sequence analysis revealed that the clone LMP131 encodes a peptide which is highly homologous to the tomato pollen-preferential gene, LAT59, which encodes a putative pectate lyase. The clone LMP134 encodes a peptide that shows an extensive similarity to a variety of thioredoxins. The third clone LMP132 encodes a 182-residue protein that has no significant homology to known sequences.     

Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.     

无核荔枝果实形成差异表达基因cDNA的克隆

采用抑制差减杂交技术(Suppression Subtractive Hybridization,SSH)分离与海南无核荔枝果实形成相关的差异表达基因的cDNA片段,为克隆相关基因提供研究基础。分别以无核荔枝的有核幼果为driver;无核幼果为tester,建立差减cDNA文库。经Reverse Northern Dot-Blot筛选该文库,共获得61个阳性克隆,随机选取17个克隆进行测序,共获得10条非重复序列,对其中较长的7个序列进行同源分析,结果表明:有6个序列在荔枝中为首次报道。