S.G. Wildman

Studies on fraction I protein IV. Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein

Abstract Tryptic peptides were resolved from the small subunit of highly purified Fraction I protein obtained from Nicotiana tabacum, Nicotiana glutinosa, Nicotiana glauca and four reciprocal, F1 hybrids: N. tabacum × N. glutinosa ; N. tabacum × N. glauca . Information for synthesis of an extra N. tabacum peptide was transferred by pollen to N. glutinosa egg cells and therefore the Mendelian mode of inheritance signifies nuclear DNA as containing the code for the primary structure of the small subunit. Two differences in peptides between N. tabacum and N. glauca were also inherited in a Mendelian manner. Transfer of the new N. tabacum information to N. glauca egg cells also supressed synthesis of the N. glauca type of Fraction I protein.

Chloroplast DNA codes for the primary structure of the large subunit of Fraction I protein

Abstract Resolution of the tryptic peptides obtained from the large subunit of Fraction I proteins isolated from Nicotiana species indigenous to Australia revealed one peptide that was not found in Nicotiana species indigenous to the Western Hemisphere. The extra peptide appeared in the reciprocal F1 hybrids N. gossei ( Australia ) × N. tabacum ( Western Hemisphere ) , only when N. gossei was the female parent. The maternal mode of inheritance requires chloroplast DNA to code for the sequence of amino acids in the large subunit, in contrast to a previous finding that nuclear DNA codes for the small subunit.

Multiple peptide composition of the large and small sub-units of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electrofocusing

Abstract 14C-labeled Fraction I protein was synthesized by Nicotiana tabacum leaves during 20 h of photosynthesis in the presence of 14CO2. The crystalline protein was carboxymethylated, dissociated by sodium dodecylsulfate and subjected to Sephadex G-100 chromatography which resolved the large and small subunits in a 3:1 ratio according to 14C content of the peptides. The large subunit migrated as 56 000 dalton monomers during sodium dodecylsulfate-polyacrylamide-gel electrophoresis; the small subunits as 12 500 dalton monomers. Fingerprinting resolved 54–56 tryptic peptides from the large subunit whereas 53–55 were expected from the number of arginine-lysine residues. Electrofocusing of carboxymethylated Fraction I protein showed the large subunit to be composed of three peptide bands which resolved in the pH 7 region and were nearly equal in density of staining. The number of tryptic peptides of the small subunit was twice the number of arginine-lysines; electrofocusing produced two approximately equal peptide bands in the pH 6 region. The same number of peptides as well as proportions of each was achieved by either staining of the bands or by autoradiography of 14C-labeled Fraction I protein. The three bands of different isoelectric points composing the large subunit have proved to be exceptionally useful as phenotypic markers for chloroplast DNA genes.

Studies on fraction I protein: II. Comparison of physical, chemical, immunological and enzymatic properties between spinach and tobacco fraction I proteins

Abstract Fraction I proteins from tobacco and spinach were homogeneous and displayed Svedberg constants (s20,w) of 18.1 ± 0.1 and 18.2 ± 0.1, respectively. Mixing the two proteins produced neither separation nor skewness in the velocity sedimentation schlieren patterns. Both sodium dodecyl sulfate and alkali caused dissociation of the two proteins into two subunits which were readily separated from each other by Sephadex column chromatography. The ratio in amount of large to small subunits was the same for both tobacco and spinach Fraction I proteins. Disruption of SS bonds was not a requirement for dissociation and separation of subunits. Immunological analyses using antibodies against Fraction I protein from tobacco or spinach showed both proteins to be entirely homogeneous antigens, although the two proteins shared cross-reactive sites. Tryptic peptide analysis showed that 17 out of 22 (spinach) or 24 (tobacco) peptides of the large subunit were alike. In marked contrast, 9 out of 15 (spinach) or 10 out of 16 (tobacco) tryptic peptides of the small subunit were distinctly different. As for ribulose diphosphate carboxylase (EC 4.1.1.39) activity, the Km for CO2 was the same for both tobacco and spinach Fraction I protein (1.7 · 10−2 M) whereas a small difference in Km for ribulose diphosphate was found (1.6 · 10−4 M for tobacco; 3.0 · 10−4 M for spinach).

The early events of infection with tobacco mosaic virus nucleic acid.

Abstract The early events of infection with strains U1 and U2 of tobacco mosaic virus, and the nucleic acids isolated therefrom, were compared in the local lesion host Nicotiana glutinosa. The technique used was to irradiate infective centers with ultraviolet light at different times after initiation of infection. Infective centers initiated by intact virus display an initial lag period during which no change in resistance to ultraviolet light occurs. This lag period is 2 1 2 ; hours for the U2 strain and 5 hours for the U1 strain. In contrast, infective centers caused by the nucleic acid isolated from these two strains behave alike and display little or no lag before they begin to increase in resistance to ultraviolet light. The conclusion is reached that when infection is initiated with the intact virus, the nucleic acid moiety of the virus is freed from the protein moiety soon after infection is established and before virus replication begins.

Polypeptide Composition of Fraction 1 Protein from Parasexual Hybrid Plants in the Genus Nicotiana

Analysis of the subunit polypeptide composition of Fraction 1 proteins gives information on the expression of both nuclear and chloroplast genomes; the large subunits of the protein are coded by chloroplast DNA, whereas the small subunits are coded by nuclear DNA. Fraction 1 protein isolated from the leaves of parasexual hybrid plants derived from the fusion of protoplasts of Nicotiana glauca and N. langsdorffii contains the small subunit polypeptides of both parent species and the large subunit polypeptides of only N. glauca. Fraction 1 protein isolated from the leaves of a hybrid plant obtained after the uptake of chloroplasts of N. suaveolens by protoplasts of white tissue of a variegating mutant of N. tabacum contains the large subunit polypeptides of both N. suaveolens and N. tabacum, as well as the small subunit polypeptides of both these species.

Identification of several chloroplast DNA genes which code for the large subunit of Nicotiana Fraction I proteins

SummaryElectrofocusing of carboxymethylated, crystalline Fraction I proteins in polyacrylamide gels containing 8 M urea resolves the large subunit into three major peptides and the small subunit into one or more peptides. Electrofocusing of proteins isolated from leaves of the reciprocal, F1, hybrids: N. glutinosa x N. tabacum, N. glauca x N. tabacum, N. glauca x N. langsdorfii and the parental species confirms that coding information for the large subunit is inherited only by the maternal line whereas both parents contribute coding information for the small subunit. The analysis shows that one or more of the three peptides of the large subunit of Fraction I proteins from different Nicotiana species differ in isoelectric point and therefore serve as phenotypic markers for chloroplast DNA genes.

Fraction 1 Protein and the Origin of Polyploid Wheats

The nature of the interspecific hybridizations giving rise to the polyploid wheats has been determinedfrom an analysis of the polypeptide compositions offraction I proteins isolatedfrom diploid, tetraploid, and hexaploid wheat species. In the origins of the tetraploid wheat, the B-genome donor provided the chloroplast genome and was therefore the maternal parent in the cross with Triticum monococcum. Subsequently, the tetraploid wheat, T. dicoccum, was the maternal parent in the cross with Aegilops squarrosa, giving rise to the hexaploid wheat, T. aestivum. Ofthe small number ofdiploid species examined, only Ae. speltoides has a chloroplast genome similar to that of the polythis method outside the genus Nicotiana we have examined the fraction I proteins of species implicated in the origins of polyploid wheat. These analyses provide some indications as to the nature of the B-genome donor and further indicate the directions of the interspecific crosses that gave rise to the tetraploid and hexaploid wheats. The wheat species examined were the hexaploid Triticum aestivum, the tetraploids T. timopheevii and T. dicoccoides and its cultivated form T. dicoccum, and the diploids Aegilops speltoides, Ae. squarrosa, T. urartu, and T. boeoticum and its cultivated form T. monococcum. Fraction 1 proteins were isolated from the supernatant fraction obtained by homogenizing 14-day-old leaves in 3 volumes of 0.05M tris-HCI buffer, pH 7.8, containing 0.2M NaCI, 0.5 mM ethylenediaminetetraacetic acid, 10 mM potassium metabisulfite, and 20 percent (weight to volume) Dowexl(x2) and then centrifuging for 60 minutes at 77,000g. Fraction 1 proteins were precipitated from the extract by ammonium sulfate at 50 percent saturation, and then extraneous proteins were removed by passage through a column of Sepharose 4B to which antibodies to fraction 1 protein had been covalently linked (7). Fraction 1 protein bound to the column was then eluted in the form of dissociated subunits by 8M urea in 0.05M tris-HCI, pH 7.8, 0.2M NaCI. The protein was then S-carboxymethylated before analysis by isoelectric focusing in the presence of 8M urea (1). As shown in Fig. 1, all fraction I proteins from the wheat species contain three large subunit polypeptides and a single small subunit polypeptide. The species fall into two groups on the basis of the isoelectric points of the large subunits. One group, having higher isoelectric points, contains T. aestivum, T. dicoccum, and Ae. speltoides, as well as T. timopheevii (not shown) and T. dicoccoides (see Fig. 2). The other group, with lower isoelectric points for the large subunit polypeptides, contains the diploid species, T. boeoticum, T. urartu, and Ae. squarrosa, as well as T. monococcum (not shown). These differences allow some conclusions to be drawn concerning the course of evolution of the polyploid wheats, because the mode of inheritance of the large subunit polypeptides of fraction I protein is the same in wheat as in the Nicotiana species. This is shown in Fig. 2 by an analysis of the polypeptide composition of fraction I proteins from reciprocal interspecific hybrids of T. boeoticum and T. dicoccoides. The large subunit polypeptides of fraction I protein from the hybrid species are inherited solely from the maternal parent, as has been demonstrated previously for interspecific hybrids in the genus Nicotiana (4).

EFFECT OF TMV MULTIPLICATION ON RNA AND PROTEIN SYNTHESIS IN TOBACCO CHLOROPLASTS.

Abstract 32 P- and 3 H-labeled amino acids were applied for 8 hours to “third” leaves of young tobacco plants during the period of very rapid TMV accumulation. Nucleoproteins and proteins in extracts of the leaves were resolved by sucrose density gradient centrifugation. Both 32 P and 3 H were found in the TMV fraction and the 80 S cytoplasmic ribosomes, but not in the chloroplast 70 S ribosomes. Furthermore, a great reduction in 3 H labeling of chloroplast proteins occurred in TMV-infected leaves. Gel electrophoresis resolution of 32 P total leaf RNAs showed that the 23 S and 16 S ribosomal RNAs of chloroplasts were not labeled whereas the 25 S and 18 S cytoplasmic ribosomal RNAs were. Isolated chloroplasts from TMV-infected leaves were found to have a reduced capacity for both nucleoside triphosphate incorporation into RNA and amino acid incorporation into protein. The ratio of AMP GMP and UMP GMP incorporation into RNA by isolated chloroplasts from TMV-infected leaves was markedly different from that by chloroplasts isolated from healthy leaves. The results indicate that chloroplasts cannot be the initial site of TMV synthesis and that during the period of grand TMV accumulation, ribosomal RNA and mRNA production from chloroplast DNA is switched off by an unknown regulator. A hypothesis is presented to account for the appearance of mosaic symptoms based on the regulation of chloroplast metabolism by TMV multiplication.

The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light.

Abstract The early events of tobacco mosaic virus infection were studied by following the radiosensitivity of infection as a function of time after inoculation. At 20°, the survival of infection as a function of ultraviolet light dose is exponential for approximately 5 hours after inoculation, after which the appearance of new intracellular infectious particles is indicated by a change to a multitarget survival curve. During the first 5 hours after inoculation, the infection is characterized by three discernible phases of sensitivity to ultraviolet light (UV): (1) a period during which the infection has essentially the same sensitivity as the virus in vitro ; (2) a period during which resistance to UV increases; and (3) a period during which a secondary level of resistance is maintained. The timing of these phases is markedly dependent on temperature. The exponential survival curve during the first phase is independent of inoculum concentration, indicating that an exclusion mechanism is operative which permits only one infectious virus particle to initiate an infection.