John G. K. Williams

[55] – Genetic Analysis Using Random Amplified Polymorphic DNA Markers

Publisher Summary The most commonly used DNA markers in genetic mapping, genetic diagnostics, molecular taxonomy, and evolutionary studies are restriction fragment length polymorphisms (RFLPs). This chapter presents the detailed experimental protocols for random amplified polymorphic DNA (RAPD) assays and applications, emphasizing their use for genetic analysis in plants. To perform a RAPD assay, a single oligonucleotide of an arbitrary DNA sequence is mixed with genomic DNA in the presence of a thermostable DNA polymerase and a suitable buffer, and then is subjected to temperature cycling conditions typical of the polymerase chain reaction. At an appropriate annealing temperature during the thermal cycle, the single primer binds to sites on opposite strands of the genomic DNA that are within an amplifiable distance of each other, and a discrete DNA segment is produced. The presence or absence of this specific product, although amplified with an arbitrary primer, will be diagnostic for the oligonucleotide-binding sites on the genomic DNA. The chapter additionally presents the statistical aspects of genetic mapping with RAPD markers.

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane‐protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin‐resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.

Sequencing and modification of psbB, the gene encoding the CP-47 protein of Photosystem II, in the cyanobacterium Synechocystis 6803

The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.

Nucleotide sequence of psbC, the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II, in the cyanobacterium Synechocystis 6803.

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.

Nucleotide Sequences of Both psbD Genes from the Cyanobacterium Synechocystis 6803

A genetic system has been developed for site-specific mutagenesis of the D2 polypeptide of Photosystem II in the cyanobacterium Synechocystis 6803. In chloroplasts, this polypeptide is encoded by the psbD gene (1). The discovery of significant amino acid homologies between the D2 polypeptide and the M-subunit protein of the bacterial photosynthetic reaction center has led to the proposal that certain amino acid residues in D2 might bind the reaction center chlorophyll, the quinone QA, and the ferrous non-heme iron.

Site-Directed Mutations of Two Histidine Residues in the D2 Protein Inactivate and Destabilize Photosystem II in the Cyanobacterium Synechocystis 6803

Site-directed mutations were created in the cyanobacterium Synechocystis 6803 to alter specific histidine residues of the photosystem II (PS II) D2 protein. In one mutant (tyr-197). the his-197 residue was replaced by tyrosine, in another mutant (asn-214), his-214 was changed into asparagine. The tyr-197 mutant did not show any low-temperature fluorescence attributable to PS II. but contained a PS II chlorophyll-protein, CP-47, in significant quantities. Another PS II chlorophyll-protein, CP-43, was absent, as was PS II-related herbicide binding. The asn-214 mutant showed a blue-shifted low-temperature fluorescence maximum around 682 nm. but did not have a significant amount of membrane-incorporated CP-43 or CP-47. Herbicide binding was also absent in this mutant. These data indicate a very important role of the his-197 and his-214 residues in the D 2 protein, and are interpreted to support the hypothesis that the D2 protein and the M subunit from the photosynthetic reaction center of purple bacteria have analogous functions. According to this hypothesis, his-197 is involved in binding of P680. and his-214 forms ligands with Qᴀ and Fe2+. In absence of a functional D2 protein, the PS II core complex appears to be destabilized as evidenced by loss of chlorophyll-proteins in the mutants.

Random amplified polymorphic DNA (RAPD) markers

Williams [14] and Welsh and McClelland [13] demonstrated the utility of single short oligonucleotide primers of arbitrary sequence for the amplification of DNA segments distributed randomly throughout the genome. Welsh and McClelland showed that the pattern of amplified bands could be used for genome fingerprinting [13] and Williams et al. [ 14] showed that the differences (polymorphisms) in the pattern of bands amplified from genetically distinct individuals behaved as mendelian genetic markers (named RAPDs, for Random Amplified Polymorphic DNA). Well-saturated maps of the Arabidopsis [11] and pine [4] genomes have been constructed using RAPD technology. A single set of arbitrary-sequence 10-mers may be used for mapping or fingerprinting any species. The many advantages of RAPD markers over RFLPs or isozyme markers accelerated the adoption of RAPD technology for the construction of genetic maps, fingerprinting, and population genetic studies [6]. Current reviews of the applications of RAPD technology are available [ 10, 12].

Genetically Engineered Cytochrome B559 Mutants of the Cyanobacterium, Synechocystis 6803

Cartridge mutagenesis procedures were used to create insertion and deletion mutations in the psbE and psbF genes encoding the cytochrome b559 protein of the photosystem II complex in the cyanobacterium, Synechocystis 6803. The PSII complexes of the mutants are inactive. However, the chlorophyll-binding CP47 and CP43 proteins are present in a deletion mutant, indicating that they are stably integrated in thylakoid membranes lacking cyt b559.

Site-Directed Mutagenesis in the Photosystem II Gene psbD, Encoding the D2 Protein

Using site-directed mutagenesis, it was shown that two histidine residues (his-197 and his-214) in the Photosystem II protein D2 play a crucial role in the function of the Photosystem II complex in the cyanobacterium Synechocystis 6803. A specific change of either histidine residue into tyrosine and asparagine, respectively, lead to a loss of PS II activity. These histidine residues have been hypothesized to be analogous to the histidine residues of the M-subunit from purple bacteria involved in binding of the reaction center chromophore, and Q and Fe , respectively (see ref. 1 and 2). The data presented here can be interpreted to support this hypothesis. The data obtained are consistent with the hypothesis that the binding determinants for the PS II reaction center are created mainly by D1 and D2.