Paul R. Whitfeld

Comparison of chloroplast DNAs by specific fragmentation with EcoRI endonuclease

SummaryA technique has been developed whereby chloroplast DNA can be digested with EcoRI endonuclease without prior isolation of the DNA from the organelle. The specific fragmentation patterns produced show that chloroplast DNAs from different families or genera of plants have few if any bands in common, and it is not until comparisons are made between species of the same genus that similarities become apparent. The number of bands in common between species varies considerably from genus to genus and the degree of similarity of chloroplast DNAs may be correlated with how closely related the species are as judged by their ability to form viable hybrids.

Occurrence of short particles in beans infected with the cowpea strain of TMV: II. Evidence that short particles contain the cistron for coat-protein

Abstract Preparations of the cowpea strain of TMV contain both large and small particles. RNA extracted from these particles has been translated in a cell-free protein synthesizing system derived from wheat germ. The polypeptide products have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by immunoprecipitation followed by SDS-PAGE and also by two-dimensional tryptic peptide mapping of the immunoprecipitated material. Coat-protein was shown to be a major product of in vitro protein synthesis directed by total viral RNA. It was synthesized in a form slightly larger than the long polypeptide of the virus capsid as judged by peptide mapping and incorporation of [35S]methionine, an amino acid not present in B-TMV coat-protein. RNA extracted from a purified preparation of short particles stimulated the wheat-germ system to synthesize coat-protein almost exclusively. It was concluded that short particles carry a cistron for coat-protein. RNA isolated from a preparation of long particles programmed the synthesis of a series of polypeptides ranging in size from 130,000 to 5000 daltons, and including some coat-protein. More extensive purification of the high molecular weight RNA showed that the synthesis of coat-protein was not attributable to this RNA but to residual low molecular weight RNA from short particles. Thus RNA from long particles resembles RNA from Ul-TMV in its inability to program a wheat-germ system to synthesize appreciable amounts of coat-protein. Evidence is given elsewhere that long particle RNA actually contains a cistron for coat-protein. Translation of long RNA could be suppressed by the addition of short RNA to the in vitro system, resulting in a polypeptide profile dominated by coat-protein. Initiation sites in the coat-protein cistron on the short RNA must therefore be preferentially recognized by the wheat-germ protein synthesizing system.

Chloroplast DNA variation in isonuclear male-sterile lines of Nicotiana

SummaryChloroplast DNAs of six isonuclear malesterile tobacco lines and their respective parental species were analysed with the restriction endonuclease, EcoR1. Four of the lines had the same fragmentation pattern as their respective maternal species. Two lines had a pattern which was different to either parental species. The results show that nucleotide changes can occur in chloroplast DNA of isonuclear male-sterile lines, and are discussed in relation to the possible involvement of chloroplast DNA in cytoplasmic male-sterility in tobacco.

Molecular cloning of two different cDNAs for maize acetyl CoA carboxylase.

Acetyl CoA carboxylase (EC 6.4.1.2) in plants is a chloroplast-localized, biotin-containing enzyme that catalyses the carboxylation of acetyl CoA to malonyl CoA, the first committed step of the fatty acid biosynthesis pathway. Acetyl CoA carboxylase is the target site for the monocotyledon-specific aryloxy-phenoxypropionate and cyclohexanedione groups of herbicides. We have purifed a herbicide-sensitive acetyl CoA carboxylase from maize leaves to homogeneity (specific activity 7 μmol min-1 mg-1), separating it during the purification from a minor herbicide-resistant acetyl CoA carboxylase. The purified enzyme is a dimer of 230 kDa subunits. Antibodies raised to the purified acetyl CoA carboxylase detected three cross-reacting clones in a maize leaf cDNA expression library, each having an insert of 4–4.5 kb. Restriction analysis and sequencing showed that the cDNAs were derived from two different transcripts. Comparison of the deduced amino acid sequences with those of chicken and yeast acetyl CoA carboxylases confirmed that both types encoded acetyl CoA carboxylase, corresponding to the C-terminal half of the enzyme. The overall identity of the maize and chicken sequences was 37% (58% similarity) but for some shorter regions was much higher. Analysis of six other acetyl CoA carboxylase clones recovered from the maize cDNA library showed four belonged to one type and two to the other. The nucleotide sequence similarity between the two types of cDNA was approximately 95% in the coding region but considerably less in the 3′-untranslated region. Northern blot analysis of maize RNA showed a single band of 8.2–8.5 kb for acetyl CoA carboxylase mRNA. Southern blot hybridisations indicated that there are probably no more than two genes in maize for acetyl CoA carboxylase. The possible significance of two different cDNAs for acetyl CoA carboxylase is discussed.

Occurrence of short particles in beans infected with the cowpea strain of TMV: I. Purification and characterization of short particles

Abstract Bean plants infected with the cowpea strain of TMV (B-TMV) have been found to contain two size-classes of virus particles. The long particles are typical TMV rods, approx 300 nm long, and contain RNA of molecular weight 2 × 10 6 . The short particles are rods, approx 40 nm long, and contain RNA of molecular weight 0.3 × 10 6 ± 20,000. Base composition analysis of the two RNA species showed minor differences in the adenylic and uridylic acid content. Electrophoresis of the coat-protein of B-TMV on 14% polyacrylamide gels in the presence of sodium dodecyl sulfate revealed the existence of two components of molecular weights approx 18,000 and 16,500. Protein derived from long particles was composed mainly of the larger component whereas protein derived from short particles was composed largely of the 16,500 molecular-weight component. In double diffusion tests, however, using antiserum prepared against a mixture of the proteins, the proteins of short and long particles gave reactions of identity. Tryptic peptide maps of the proteins from the short and from the long particles confirmed that the two proteins were basically the same, the smaller component apparently lacking at least one peptide present in the 18,000 molecular-weight component. Short and long particles were also found in association with B-TMV infections of tobacco plants. Short particles could not be detected in tobacco plants infected with the common strain of TMV. The ratio of the number of short particles to the number of long particles was 1:1 to 2:1 for bean or tobacco plants which had been inoculated 4–6 weeks previously. Purified RNA from short particles (0.3 × 10 6 daltons) was not infectious when inoculated onto bean plants. Long particle RNA, purified through sucrose and formamide-sucrose density gradients, produced symptoms on inoculated bean plants typical of B-TMV infections. Virus preparations extracted from such plants contained short as well as long particles. The possible significance of the short particles in virus infection is discussed in the light of the observation that the short RNA contains the cistron for the viral coat-protein.

Differential recognition of chloroplast and cytoplasmic messenger RNA by 70S and 80S ribosomal systems.

Chloroplasts are known to contain DNA and to be competent to express the genetic information contained in that DNA. They have their own DNAdependent RNA polymerase and also their own protein-synthesizing apparatus [l] . The latter is a 70s ribosomal system, similar in many respects to that of prokaryotes but quite distinct from the 80s ribosomal system of the plant cytoplasm. In this report we show that it is possible to differentiate between the messenger RNA in the chloroplast and that present in the cytoplasm. Chloroplast mRNA can be translated by a 70s ribosomal, proteinsynthesizing system from E. coli (E. coli S30) but not by an 80s ribosomal system from wheat germ (wheat germ S30), whereas mRNA in the cytoplasm can be translated by a wheat germ S30 but not by an E. coli S30.

Size distribution and In vitro translation of the RNAs isolated from turnip yellow mosaic virus nucleoproteins

Abstract Purified preparations of TYMV contain a number of minor nucleoprotein components distinguishable on the basis of their density in CsCl gradients (R. E. F. Matthews, Virology 12 , 521–539, 1960). When analysed by polyacrylamide-gel eletrophoresis, the RNA from each of these various components was found to consist of a characteristic set of molecular-weight species. Full-size RNA (2 × 10 6 daltons) was present only in nucleoproteins B 1 and B 2 . Nucleoproteins B 0 , B 00 , and B 000 contained RNAs ranging in size from approx 1.3 to 0.28 × 10 6 daltons. The 0.28 × 10 6 -dalton RNA species was detectable in all nucleoprotein fractions, but was a significant proportion of the RNAs of B 000 and B 00 . Translation of the RNA from each of the nucleoproteins in the wheat germ cell-free protein synthesizing system yielded essentially similar patterns of polypeptides which ranged in size from 5000 to 70,000 daltons. The major radioactive product migrated on SDS-polyacrylamide gels to the same position as TYMV coat protein. The data suggest that the 0.28 × 10 6 -dalton RNA component is the cistron for coat protein, and that it and other RNAs associated with TYMV infection are encapsidated.

The γ-subunit of spinach chloroplast ATP synthase: isolation and characterisation of cDNA and genomic clones

AbstractcDNA and genomic clones encoding the complete precursor polypeptide of the γ-subunit of spinach chloroplast ATP synthase have been isolated and characterised. The longest cDNA (1320 bp), selected from a λgt 11 spinach cDNA library, encoded a 364 amino acid residue protein (Mr 40 028) that included a putative 41 residue N-terminal transit peptide. All the γ-subunit cDNAs analysed were derived from the same gene and Southern blot analysis ofBam HI restricted spinach DNA showed only one hybridising band (ca. 15 kb). Analysis of the cloned 15 kb genomic fragment, selected from a λEMBL4 spinach DNA library with a cDNA probe, confirmed there was a single gene for the γ-subunit. Sequencing, primer extension and northern blot analysis showed the gene contains two introns, 1066 and 665 bp in length, a 173 bp 5′ untranslated region and a 213 bp 3′ untranslated region. A 1450 nucleotide γ-subunit transcript was detected in RNA from light-grown and dark-grown spinach.

Characterization of the spinach chloroplast genes for the S4 ribosomal protein, tRNAThr (UGU) and tRNASer (GGA)

SummaryThe map location and nucleotide sequence of the genes for the S4 ribosomal protein (rps4) and for tRNAThr (UGU) (trnT) and tRNASer (GGA) (trnS) on spinach chloroplast DNA have been determined. rps4 lies approximately 5 kb 3′ to atpBE in the large single copy region and is transcribed in the same direction as atpBE. It has a 178 bp leader sequence, a 603 bp coding region and 620 bp 3′ tail. The sequence of the coding region is 83% homologous with that of maize rps4 (29) and the deduced amino acid sequences from the two species are 7% homologous. The spinach and Escherichia coli S4 proteins are only 36% homologous. As in the case of maize, trnT lies upstream from and on the same strand as rps4 whereas trnS lies downstream and on the opposite strand. Transcription of rps4 apparently proceeds past trnS.

Organization and Structure of the Genes for the β and ε Subunits of Spinach and Pea Chloroplast ATPase

Chloroplast ATPase (CF1) is composed of five subunits, three of which (α,β and e) are synthesized within the chloroplast (see (1) for references) and coded by chloroplast genes (2). The γ and δ subunits are synthesized in the cytoplasm and are presumed to be coded by nuclear genes (see (1) for references). The genes for the β and e subunits map close to the gene (rbcL) for the large subunit of ribulose bisphosphate carboxylase but the gene for the α subunit is located some 40 kb away on the spinach chloroplast DNA (cpDNA) map (2). In this paper we report the results of our investigations into the structure and organization of the genes for the β and e subunits of ATPase from Spinacia oleracea and Pisum sativum chloroplasts.