Anthony R. Cashmore

Light-inducible and tissue-specific expression of a chimaeric gene under control of the 5'-flanking sequence of a pea chlorophyll a/b-binding protein gene

We have investigated the regulatory functions of the 5′‐flanking sequences of a chlorophyll a/b‐binding protein gene from Pisum sativum, using the neomycin phosphotransferase (II) activity from Tn5 as an enzymatic reporter. We show that 0.4 kb of the upstream flanking sequences of this gene are sufficient for both organ‐specific and light‐regulated expression of our chimaeric constructs in transformed tobacco plants. In addition, we show that sequences farther upstream have a significant influence on the level of transcription of these constructions.

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the gamma Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phase contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAb genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.

Expression of nuclear and plastid genes for photosynthesis-specific proteins during tomato fruit development and ripening.

SummaryThe expression of plastid and nuclear genes coding for photosynthesis-specific proteins has been studied during tomato fruit formation. The steady-state transcript levels for the large (rbcL) and small (rbcS) subunit of RuBPC/Oase, as well as the thylakoid membrane proteins, the 32 kD QB-binding protein of PS II (psbA), the P700 reaction center protein of PS I (psaA) and the chlorophyll a/b-binding protein (cab) vary at different time points during fruit development and ripening. Messenger RNA levels of plastid-encoded photosynthesis-specific genes (rbcL, psbA) are at least several fold higher, relative to respective nuclear-encoded genes (rbcS, cab). The transcript levels for the large and small subunit of RuBPC/Oase are highest in approximately 14-day-old tomato fruits, while the chl a/b-binding protein, the P700 reaction center protein and the 32 kD QB-binding protein reach their maxima in approximately 7-, 14- and 25-day-old tomato fruits, respectively. The inactivation of the photosynthesis-specific genes occurs during the first period of fruit formation. In addition, there is considerable variation in the mRNA levels of these photosynthesis-specific genes in four organs of tomato (leaves, fruits, stems, roots).

The Lucretian swerve: The biological basis of human behavior and the criminal justice system

It is widely believed, at least in scientific circles, that living systems, including mankind, obey the natural physical laws. However, it is also commonly accepted that man has the capacity to make “free” conscious decisions that do not simply reflect the chemical makeup of the individual at the time of decision—this chemical makeup reflecting both the genetic and environmental history and a degree of stochasticism. Whereas philosophers have discussed for centuries the apparent lack of a causal component for free will, many biologists still seem to be remarkably at ease with this notion of free will; and furthermore, our judicial system is based on such a belief. It is the author’s contention that a belief in free will is nothing other than a continuing belief in vitalism—something biologists proudly believe they discarded well over 100 years ago.

In Vitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex

Recent studies have established that transport across chloroplast envelopes of proteins which are synthesized by cytoplasmic ribosomes can occur by a post-translational mechanism (1,2). Dobberstein et al. (3) first discovered that a major chloroplast stromal protein, the small subunit (S) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) is synthesized by free polysomes in the green alga, Chlamydomonas reinhardtii. Moreover, they found that translation of the small subunit messenger RNA in vitro yields a precursor (pS) 4000–5000 daltons larger than the mature protein. Upon incubation with a cell-free Chlamydomonas extract pS can be processed to the mature form and a small peptide fragment designated the transit peptide (4,5). Dobberstein et al. (3) proposed that transport of the RuBPCase small subunit in vivo occurs after it is completely synthesized and that the transit sequence on pS facilitates its post-translational interaction with the chloroplast envelope. This proposed mechanism is fundamentally distinct from the co-translational transport across endoplasmic reticulum membranes of proteins which are synthesized by membrane-bound ribosomes (6,7). Precursor forms of the RuBPCase small subunit also have been found among the translation products of spinach (1), pea (1,2,8) and duckweed (9) mRNA in cell-free systems.

Targeting Nuclear Gene Products into Chloroplasts

Plant cells contain several distinct subcellular compartments or organelles that perform specialized functions within the cell. Two of these organelles, chloroplasts and mitochondria, exist in a semi-autonomous state within the cell containing their own genetic system and protein synthesis machinery. However, the majority of chloroplast and mitochondrial proteins are nuclear encoded, synthesized on free cytosolic ribosomes and then imported into their respective organelles. Many of these proteins are synthesized as higher molecular weight precursors that are processed to their mature forms either during or shortly after import. The proper targeting and import of these polypeptides depends on a transit peptide located at the amino-terminus of the precursor (for recent reviews see Ellis, 1981; Cashmore et al., 1985; Schmidt and Mishkind, 1986; Hay et al., 1984 and Hurt and van Loon, 1986).

Reply to Hinsen: Free will, vitalism, and distinguishing cause from effect

Hinsen’s conclusion that a “scientific model for free will is impossible” may alarm many believers in free will (1). However, rather than abandoning the concept, which would be the standard scientific procedure in the absence of any model, Hinsen notes that a pragmatically minded person would argue, “we should trust our perception, which tells us that we do have free will.” I argue that our belief in free will is largely dependent on our observations that thought processes correlate with behavior (2). As scientists, we know that correlations tell us nothing about causality, and in this particular case, the correlation is simply that. Indeed, what little evidence there is addressing the causal relationship between conscious thought and behavior suggests that the former is simply the effect of neural activity, not its cause (see references cited in ref. 2). For these reasons I suggested that it was appropriate to discard the belief in free will, at least until someone could provide an appropriate testable molecular model (2–4).

The Characterisation of Leaf Messenger RNAs and their Use in the Synthesis of Complementary DNAs

The major products of cytoplasmic protein synthesis in pea leaves correspond to polypeptide components of two chloroplast proteins. These polypeptides are the small subunit of ribulose-1,5- bisphosphate (RuBP) carboxylase and the constituent polypeptides of the chlorophyll-protein complex II (CPII) or light-harvesting chlorophyll a/b protein (Cashmore, 1976 and Schmidt et al., this volume). Both the small subunit and the CPII polypeptides are synthesized, on free cytoplasmic polyribosomes, as soluble precursors which function in the post-translational transport of the polypeptides from their site of synthesis into the chloroplast (Dobberstein et al., 1977; Cashmore et al., 1978; Highfield and Ellis, 1978; Apel and Kloppstech, 1978; Chua and Schmidt, 1978; Schmidt et al., this volume). From studies on the mode of inheritance of peptide variants it appears that these cytoplasmically synthesized polypeptides are encoded by nuclear genes (Kawashima and Wildman, 1972; Kung et al., 1972). In contrast, the large subunit of RuBP carboxylase is translated on chloroplast ribosomes (Blair and Ellis, 1973) and is encoded by the chloroplast genome (Coen et al., 1977).