The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.
Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.
SUMMARYA library of cDNA clones was produced from the approximately 6 kb RNA of the MAV isolate of barley yellow dwarf virus (BYDV) in bacteriophage 2gt l 1, by a method that involved random priming and cloning ds cDNAs of between 1.0 and 2-5 kbp. Screening with antiserum to the dissociated coat protein of the MAV isolate showed that approximately 2.5 % of the recombinants were capable of expressing this protein.After subcloning some inserts into the plasmid pUC18, restriction endonuclease mapping showed that they collectively represented at least 85% (a total of 5-1 kbp) of the BYDV genome. We did not attempt to determine which, if any, of the immunologically positive clones expressed the entire coat protein, but of the nine examined, all shared a region of approximately 1000 bp, located between 750 bp and 1750 bp from the 3' terminus of the restriction map. Sequence homology among different isolates of BYDV was examined by using selected MAV cDNA clones as probes in viral nucleic acid hybridization studies. Hybridization specificity varied according to the origin of the clones within the BYDV genome. Those from the putative coat protein-coding region hybridized well only to the homologous MAV isolate; those from elsewhere hybridized also with another isolate from the same subgroup (P-PAV). No clones hybridized significantly to a third isolate (RPV), which is in another subgroup of BYDV. The sensitivity of detection was related to probe size; the larger clones detected as little as 70 pg of purified virus (1.4 ng/ml in a 50 Ixl sample), and with these the sensitivity of virus detection in plant extracts by dot-blot hybridization was greater than that of ELISA. INTRODUCTIONBarley yellow dwarf virus (BYDV), the type member of the luteovirus group (Matthews, 1982), comprises a cluster of interrelated viruses or viral strains that are obligately aphidtransmitted in the circulative manner. These viruses infect a very wide range of monocotyledonous hosts and collectively are regarded as the most economically important viral pathogens of small grains world-wide (Jones & Clifford, 1983). Isolates have been divided into five major types, with vector specificity as the major distinguishing characteristic (Rochow, 1970(Rochow, , 1979. This division is exemplified by the five isolates described by Rochow (Rochow, 1970(Rochow, , 1979, namely MAV specifically transmitted by Macrosiphum (=Sitobion) avenae, RPV specifically transmitted by Rhopalosiphum padi, PAV transmitted by both these vectors, RMV specifically transmitted by R. maidis, and SGV specifically transmitted by Schizaphis graminum. Furthermore, based on serology and other criteria, isolates may be grouped into two subgroups,
A maize zein cDNA clone was used to synthesize mRNA with the SP6 in vitro transcription system. Although we obtained full-length transcripts of the cDNA sequence, these were inefficient templates for protein synthesis. Removal of the 5' oligo(G) sequence that was synthesized during the cDNA cloning procedure allowed efficient translation of the mRNAs in a wheat germ cell-free protein synthesis system or in Xenopus laevis oocytes. The alteration in translational efficiency did not result from an interaction of the 5' oligo(G) homopolymer tail with the 3' oligo(C) sequence, as transcripts with or without the oligo(C) tail were translated similarly in both protein synthesis systems. Ribosome interaction with the mRNA may be affected due either to the secondary structure of the oligo(G) sequence itself, or an unusual secondary structure between the oligo(G) sequence and another region in the mRNA. Synthetic oligonucleotides at the 5' end of cloned cDNA sequences may generally be inhibitory for translation of mRNAs transcribed in vitro.
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