A barley gene (rsh1) encoding a ribonuclease S-like homologue specifically expressed in young light-grown leaves.

A group of frequent cDNA clones from a young-leaf cDNA library was found to code for a homologue of S-ribonucleases (S-RNases) involved in gametophytic incompatibility and the so-called S-like RNases active in flowers and in vegetative tissues. The derived amino acid sequence starts with a signal peptide and has a 27-amino-acid C-terminal extension of unknown function. The barley (Hordeum vulgare L.) gene, rsh1 (for RNase S-like homologue) corresponding to the cDNA clones was isolated. The gene has three introns and the position of one intron corresponds to the site of the single, small intron in the S-RNase genes. The deduced amino acid sequence of mature RSH1 shares 35% identical and 58% similar amino acid residues with an S-like RNase from tomato, RNase LE. However, two active-site histidine residues, conserved between all S and S-like RNases are replaced by serine residues in RSH1. The new barley RNase S-like homologue is clearly related to the family of active RNases but is probably not active as an RNase. Sequences from the same class of presumably inactive RNases have been recorded in maize, rice and sorghum. The barley gene is exclusively expressed in young leaf tissue and is substantially induced by light.

DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare).

The homeodomain of the knotted classes of transcription factors from plants differs from the well characterized Antp/En type homeodomains from Drosophila at key amino acid residues contributing to the DNA binding. A cDNA, Hvh21, derived from the hooded gene and encoding a full length homolog of knotted1 from maize was isolated from barley seedlings and expressed as a maltose binding protein fusion in E. coli. The purified HvH21-fusion protein selected DNA fragments with 1-3 copies of the sequence TGAC. Gel shift experiments showed that the TGAC element was required for binding and the results further indicate that the HvH21-fusion protein binds DNA as a monomer.

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles.

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/protease inhibitor, Mundy and Rogers 1986, Planta 169, 51-63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60-63% homologous to rice aleurone LTP and maize seed/coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.

In vitro binding of nuclear proteins to the barley plastocyanin gene promoter region.

Plastocyanin is a nuclear-encoded chloroplast protein participating in electron transport during photosynthesis. The plastocyanin gene is expressed in photosynthetic tissue in a developmentally regulated manner and the expression is stimulated by light. A genomic clone encoding the plastocyanin precursor was isolated from a barley (Hordeum vulgare) lambda library using a barley cDNA clone as a probe and the sequence of a 1.9-kb DNA fragment containing the plastocyanin gene was determined. TATA and CCAAT boxes are located 34-bp and 68-bp, respectively, upstream of the transcription start site, the 5'-untranslated leader is 78 nucleotides long, and the intronless gene has at least two different polyadenylation sites. DNA sites in the plastocyanin gene that mediate binding of barley nuclear proteins were mapped by mobility-shift assays with fragments of the promoter/upstream region. Two of the three specific binding sites characterised in more detail were found to form complexes with the same factor in cross-competition experiments. One of these sites, narrowed down to a 17-bp sequence at position -512, contains the consensus binding site for Myb-like transcription factors. The third specific binding site, located at position -622, contains the sequence CACGTG which is a high-affinity-binding site for transcription factors of the basic-region leucine-zipper family.

A small gene family in barley encodes ribosomal proteins homologous to yeast YL17 and L22 from archaebacteria, eubacteria, and chloroplasts.

The amino acid sequences of two barley ribosomal proteins, termed HvL17-1 and HvL17-2, were decoded from green leaf cDNA clones. The N-terminal sequences of the derived barley proteins are 48% identical to the N-terminal amino acid sequence of protein YL17 from the large subunit of yeast cytoplasmic ribosomes. Via archaebacterial ribosomal proteins this homology extends to ribosomal protein L22 from eubacteria and chloroplast. Barley L17, and ribosomal proteins L22 and L23 from the archaebacteria Halobacterium halobium and H. marismortui, are 25-33% identical. Interestingly, the barley and archaebacterial proteins share a long, central stretch of amino acids, which is absent in the corresponding proteins from eubacteria and chloroplasts. Barley L17 proteins are encoded by a small gene family with probably only two members, represented by the cDNA clones encoding HvL17-1 and HvL17-2. Both these genes are active in green leaf cells. The expression of the L17 genes in different parts of the 7-day old barley seedlings was analyzed by semiquantitative hybridization. The level of L17 mRNA is high in meristematic and young cells found in the leaf base and root tip. In the leaf, the L17 mRNA level rapidly decreases with increasing cell age, and in older root cells this mRNA is undetectable.

Two ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley.

Ubiquitin (Ubi) genes encode two types of fusion proteins: polyUbi with a varying number of direct repeats of Ubi, and Ubi-tail fusions with long or short basic C-terminal extensions. A barley (Hordeum vulgare) genomic clone has been isolated with two very similar, intronless genes encoding monoUbi-long-tail fusion peptides. The genes are arranged as direct repeats separated by 3 kb of DNA and account for two of the probable three long-tail genes in the haploid barley genome. Both genes are active and give rise to messengers about 800 nt long. The sequence of the encoded Ubi moieties is identical to the sequence of Ubi repeats of polyUbi precursors from barley and other plants. The basic tails of the peptides are 79 aa long and 71-72% homologous to corresponding sequences from yeast and man. Recently, it was found that the long and short tails are ribosomal proteins in yeast [Finley et al., Nature 338 (1989) 394-401] and the evolutionary conservation of the structure of the Ubi-tail fusion genes suggests that they serve the same function in plants. The similarity between yeast and barley Ubi-long-tail fusion genes may extend to the regulatory regions, since upstream activating sites characteristic of ribosomal protein-encoding genes in yeast (UASrpg) were found in the barley genes.

Thionin genes specifically expressed in barley leaves.

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.

Structure and expression of ubiquitin genes in higher plants.

cDNA clones encoding ubiquitin were isolated from a barley leaf cDNA library using a mammalian ubiquitin cDNA clone as probe. The nucleotide sequence of one of the clones codes for 2.2 perfect repeats of the 76-amino-acid-long ubiquitin protein with an extra lysine residue at the C-terminus. The barley ubiquitin amino acid sequence differs from the animal sequence at three positions and from the yeast sequence at two positions. The ubiquitin poly(precursor) are coded by a multigene family with 8-10 genes that produce four or five different size messengers between 700 and 2000 nucleotides in length. The large poly(A)-rich RNAs are constitutively expressed in vegetative tissues whereas the 700-nucleotide messenger(s) were only detected in tissues containing dividing cells.

Construction and characterization of a tufA-lacZ fusion coding for an E. coli EF-Tu-beta-galactosidase chimeric protein.

A new phage lambda cloning vector was constructed that has a single EcoRI site upstream from weakly expressed lacI-Z gene isolated by Müller-Hill and Kania (1974). An EcoRI fragment containing the complete tufA gene of E. coli was cloned on the vector and the recombinant phage was crossed into the str operon that has tufA as its last gene. Subsequent selection gave rise to a tufA-lacZ fusion that codes for a chimeric peptide. The fused peptide has a molecular weight of 148,000 and contains 40% of the N-terminal of EF-Tu followed by part of the lac repressor-beta-galactosidase fusion. The specific activity of the fused peptide is about half of the activity of normal beta-galactosidase.

Rates of growth, ribosome synthesis and elongation factor synthesis in a tufA defective strain of E. coli.

A tufA defective strain of E. coli was isolated which by a single deletion event acquired a tufA-lacZ fusion gene and lost the normal functional tufA gene (see accompanying paper). A correlation between the growth rate of protein synthesis was decreased to about 50% in the tufA defective strain whereas the number of EF-Tu molecules per ribosome was about 80% compared to a normal strain. The results indicate that tufB gene expression was preferentially stimulated in the tufA defective strain but the increased EF-TuB synthesis was not sufficient to make up for the loss of normal EF-TuA synthesis. Introduction of a plasmid that carries a complete tufA gene and the preceeding fusA gene but not the str-promotor into the tufA defective strain did not alleviate the slow growth or low rate of EF-Tu synthesis showing that the high rate of EF-TuA synthesis compared to the other proteins in the str operon is not augmented by a strong second promotor for the tufA gene. The tufA-lacZ fusion which takes the place of the normal tufA gene was expressed at a high rate and the beta-galactosidase activity increased with the growth rate as expected.

The complete amino-acid sequence of elongation factor Tu from Escherichia coli.

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at positon 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study.

Post-transciptional control of coordinated ribosomal protein synthesis in Escherichia coli.

The synthesis of the approximately 50 different ribosomal proteins (r proteins) is very well coordinated in Escherichia coli. As the r-protein genes are arranged in many different operons, placed at separate locations on the E. coli chromosome, it is not obvious how this coordination is maintained. The first indication that special controls are involved in the coordination came from the observations that merodiploid strains containing F' factors with some of the ribosomal genes also synthesise the r proteins in balanced amounts. The overall regulation of r-protein synthesis in reponse to changes in growth conditions is primarily mediated by changes in the rate of transcription of the r-protein genes. To investigate whether the gene dosage control seen in merodiploid strains is also transcriptional in nature or whether other mechanisms are involved, we compared the transcriptionof r-protein mRNA in haploid and merodiploid strains. It was found that the rate of transcription of the r-protein mRNA from the str-spc cluster of genes changes in proportion to the gene dosage and it is concluded that the expression of r-protein genes is adjusted by post-transcriptional control.

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.