Carl A. Price

Synthesis of coupling factor CF1 protein by isolated spinach chloroplasts.

It has been demonstrated in several laboratories that isolated chloroplasts incorporate labeled amino acids into at least nine discrete polypeptides, distinguishable by SDS-polyacrylamide gel electrophoresis, and are therefore capable of synthesizing certain of their own proteins [1-5] . The labeled polypeptides were found to be distributed among the soluble proteins, the thylakoid membrane fractions [1,2,3,5], and the envelope membranes [4,5]. Blair and Ellis [1] have identified the major labeled soluble polypeptide with the large subunit of ribulose diphosphate carboxylase. The identities of the other labeled polypeptides remain unknown. We have examined the possible synthesis of chloroplast coupling factor CFI by spinach chloroplasts through the use of a procedure developed by Strotmann et al. [6] for the preferential extraction of coupling factor from spinach chloroplasts. Our results show that, contrary to previous indications [2], the two largest subunits of coupling factor CF~ and possibly the smallest inhibitory subunit are made in this organelle.

Factors affecting the separation of photosynthetically competent chloroplasts in gradients of silica sols

Abstract Chloroplasts from spinach (Spinacia oleracea L.) can be resolved by isopycnic centrifugation in density gradients of the silica sol Ludox AM into two zones, one containing the intact and the other the stripped Chloroplasts. Factors affecting the separation and the photosynthetic activity of the intact chloroplasts recovered from the gradients were investigated and a standard set of conditions proposed. The anomalous influence of polyethylene glycols on the banding densities of both stripped and intact chloroplasts was studied and the observed “density shifts” discussed in terms of their possible cause and significance. The general method was scaled up by means of continuous-flow zonal centrifugation to prepare intact chloroplasts in amounts corresponding to tens of milligrams of chlorophyll.

Organization of the psbE, psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome

SummaryThe genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE — 8 by spacer —psbF — 110 by spacer — orf38 — 87 by spacer — orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T > A > G > C in the mRNA-like strand and group II-like boundary sequences at their 3′ and 5′ ends having the consensus 5′-GTGTG .. INTRON .. TTAATTTNAT-3′. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE psbF orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.

Organization of ribosomal protein genes rp123, rp12, rps19, rp122 and rps3 on the Euglena gracilis chloroplast genome

SummaryThe nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rp123 — 150 by spacer — rpl2 — 59 by spacer —rps19 — 110 by spacer — rp122 — 630 by spacer — rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rp123, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rp12 and rp122 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53–76% T, 25–34% A, 3–10% G and 2–7% C in the mRNA-like strand. These six introns show minimal resemblance to group IT chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5′ and 3′ ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rpsl9 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18–50%, and 29–58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rp123 and downstream from rps3.

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns

SummaryWe have determined the complete nucleotide sequence of the two genes encoding the P700 chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T > A > G > C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5′ GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3′ and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains.

[16] Isolation of plastids in density gradients of percoll and other silica sols

Publisher Summary This chapter discusses methods for the isolation of plastids in density gradients of Percoll and other silica sols. The most commonly used silica sols are Percoll, which is quite expensive, and Ludox HS and Ludox AM, which are very cheap. Silica sol gradients are generally useful for the isolation of different kinds of plastids from a wide variety of higher plants, including mesophyll and bundle sheath chloroplasts and heat-bleached chloroplasts, and from at least three kinds of algae. Separation in silica sol gradients has become the method of choice for the study of plastid composition, for the isolation of plastids other than chloroplasts, and to reisolate plastids from a complex incubation mixture where it is necessary. Plastids recovered from silica sol gradients have been used for the isolation of plastid DNA from spinach, tobacco, pepper, and Cyanophora paradoxas. Gradient-purified plastids have also been used for the studies on photosynthesis and on salt and calmodulin contents of chloroplasts.

Restriction and gene maps of plastid DNA from Capsicum annuum

SummaryChloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to chromoplasts in Capsicum annuum does not involve any large rearrangements of the plastid chromosome. A restriction map of Capsicum annuum plastid DNA was constructed using the same restriction enzymes in single digests and in all possible pair combinations. Overlapping restriction fragments were identified by digesting each product of a single digest with each of the other three enzymes. The resulting restriction map is similar to that of chloroplast DNA from other members of the Solanaceae with respect to most restriction sites. The genome size corresponds to 143 kbp. The locations of 24 genes, coding for ribosomal RNAs and for proteins of Photosystem I (PSI), Photosystem II (PSII), ATP synthase, cytochromes, the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (E.C. 4.1.1.29) (RuBPC), and ribosomal proteins were determined by probing Southern blots of Capsicum chloroplast DNA with probes of genes from spinach and tobacco. The gene locations are completely conserved with respect to those of other members of the Solanaceae and the majority of higher land plants.

Occurrence of the chromoplast protein ChrA correlates with a fruit-color gene in Capsicum annuum

Plant geneticists have determined that the color of ripe fruits of sweet peppers (Capsicum annuum L.) is determined by four genes: y, c1, c2and cl. We have compared the electrophoretic behavior of chromoplast membrane proteins of seven varieties of C. annuum which differ in these genes. ChrA was detected only in the varieties that had a y+genotype, and was not affected by variations in the other three genes. The identity of ChrA was verified by probing blots of SDS gels with antiserum to ChrA. The second known chromoplast-specific protein, ChrB, was found to be independent of all four genes. No proteins correlating with c1, c2or cl were detected in either one- or two-dimensional gels.

Plastid transcripts in chloroplasts and chromoplasts of capsicum annuum

SummaryPlastids from red and green fruits of the bell pepper, Capsicum annuum L., var. Emerald Giant, were examined for the presence of chromoplast-specific transcripts. Plastid DNA fragments produced by the restriction endonuclease PvuII were individually cut into small pieces, resolved on agarose gels, blotted, and probed with labelled cDNAs prepared from total chloroplast or chromoplast RNA. Although many chloroplast transcripts were detected at high levels in the red fruits, we saw no consistent evidence of chromoplast-specific transcripts. In order to increase the signal-to-noise ratio, chromoplast cDNA probes were absorbed with an excess of chloroplast RNAs. We also examined in greater detail regions of the plastid genome thought to contain unidentified open reading frames. None of the Southern blots provided unambiguous evidence of chromoplast-specific transcripts. Taken together with other data, these results make it unlikely, but do not exclude the possibility that plastid genes contribute to chromoplast development.

Protein synthesis by developing plastids isolated from Euglena gracilis.

Euglena gracilis has been widely used as a model for the study of light-induced plastid development [ 11. When cells that have been grown in the dark are exposed to light, the proplastids differentiate rapidly into mature, functional chloroplasts. Although the concept that plastid and nuclear genomes share control of chloroplast development has become widely accepted, there is little direct information on the contributions of the plastid itself to this process [2-71. The development of a method for the isolation from Euglena of chloroplasts capable of rapid protein synthesis [8,9] encouraged us to investigate the expression of plastid genes by examining the products of protein synthesis in organello over the course of lightinduced development. In addition to finding increases in the synthesis of certain polypeptides (as observed in [3,5,10]) we find a number of high Mr polypeptides which are synthesized transiently during early stages of development.