Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga, Volvox carteri, specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas, and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both indicative of cooption into the sexual cycle. Thus, sex-determining loci impact the evolution of both sex-related and non-sex-related genes.

Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.cytoskeleton | calcium-signaling | carbohydrate-active enzymes | stress tolerance | vitamin B 12T he red algae are one of the founding groups of photosynthetic eukaryotes (Archaeplastida) and among the few multicellular lineages within Eukarya. A red algal plastid, acquired through secondary endosymbiosis, supports carbon fixation, fatty acid synthesis, and other metabolic needs in many other algal groups in ways that are consequential. For example, diatoms and haptophytes have strong biogeochemical effects; apicomplexans cause human disease (e.g., malaria); and dinoflagellates include both coral symbionts and toxin-producing "red tides" (1). The evolutionary processes that produced the Archaeplastida and secondary algal lineages remain under investigation (2-5), but it is clear that both nuclear and plastid genes from the ancestral red algae have contributed dramatically to broader eukaryotic evolution and diversity. Consequently, the imprint of red algal metabolism on the Earth's climate system, aquatic foodwebs, and

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZ genes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type (WT). Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2-1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZ triple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

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Bacteria and chloroplasts require the ring-forming cytoskeletal protein FtsZ for division. Although bacteria accomplish division with a single FtsZ, plant chloroplasts require two FtsZ types for division, FtsZ1 and FtsZ2. These proteins colocalize to a mid-plastid Z ring, but their biochemical relationship is poorly understood. We investigated the in vitro behavior of recombinant FtsZ1 and FtsZ2 separately and together. Both proteins bind and hydrolyze GTP, although GTPase activities are low compared with the activity of Escherichia coli FtsZ. Each protein undergoes GTP-dependent assembly into thin protofilaments in the presence of calcium as a stabilizing agent, similar to bacterial FtsZ. In contrast, when mixed without calcium, FtsZ1 and FtsZ2 exhibit slightly elevated GTPase activity and coassembly into extensively bundled protofilaments. Coassembly is enhanced by FtsZ1, suggesting that it promotes lateral interactions between protofilaments. Experiments with GTPase-deficient mutants reveal that FtsZ1 and FtsZ2 form heteropolymers. Maximum coassembly occurs in reactions containing equimolar FtsZ1 and FtsZ2, but significant coassembly occurs at other stoichiometries. The FtsZ1:FtsZ2 ratio in coassembled structures mirrors their input ratio, suggesting plasticity in protofilament and/or bundle composition. This behavior contrasts with that of ␣-and ␤-tubulin and the bacterial tubulin-like proteins BtubA and BtubB, which coassemble in a strict 1:1 stoichiometry. Our findings raise the possibility that plasticity in FtsZ filament composition and heteropolymerization-induced bundling could have been a driving force for the coevolution of FtsZ1 and FtsZ2 in the green lineage, perhaps arising from an enhanced capacity for the regulation of Z ring composition and activity in vivo.Cell division in bacteria and chloroplast division in plants both require FtsZ, a tubulin-like GTPase that functions as a contractile ring, the Z ring, inside the cell or chloroplast. Mutations in bacterial FtsZ genes disrupt cell division, resulting in long filamented cells (reviewed in Refs. 1-3). Purified bacterial FtsZ undergoes GTP-dependent, hydrolysis-independent polymerization primarily into single protofilaments (4, 5), but protofilament sheets and bundles form in the presence of stabilizing agents that promote lateral interactions (6 -8). Polymerization stimulates FtsZ GTPase activity because the catalytic site for GTP hydrolysis lies in the longitudinal interface between adjacent monomers within the polymer (9). GTP hydrolysis destabilizes protofilaments, leading to disassembly (10). The in vivo structure of the bacterial Z ring is not yet clear, but recent models suggest it may be built from short overlapping protofilaments that are stabilized at the division site by accessory factors (11, 12). The Z ring functions partly as a scaffold for other cell division proteins and recently has also been shown to provide contractile force for membrane constriction (13,14).In most bacteria, including the cyanobacterial relatives of chloropl...