Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, a and f, of PC from the blue-green alga Agmenellum quadruplicatum In most photosynthetic organisms the major light-harvesting pigments are cyclic tetrapyrroles bound noncovalently to polypeptides intrinsic to the photosynthetic membrane. Blue-green algae (cyanobacteria) and red algae follow this pattern but, in addition, have large amounts of antenna pigments in the form of linear tetrapyrroles covalently bound to water-soluble polypeptides. These proteins, termed phycobiliproteins, occur as three major types: phycoerythrin, phycocyanin (PC), and allophycocyanin (1). Each phycobiliprotein consists of two subunits, a and /3, which contain characteristic numbers and types of chromophores. Phycobiliproteins aggregate to form complexes called phycobilisomes (1). The assembly of phycobilisomes is mediated by nonpigmented linker polypeptides. These linkers also alter the spectral properties of phycobiliproteins so as to ensure an efficient transfer of absorbed light energy to the membrane (2). Phycobilisomes, in turn, are found attached to the outer surface of the thylakoid membrane, perhaps in association with photosystem 11 (3).Our aim is to describe the structure of the phycobilisome and the regulation of the genes encoding its components.These studies may also shed light on the evolution of Ohycobiliprotein genes. As a first step, we have cloned and sequenced the genes encoding the a and ,8 subunit apoproteins of PC from the blue-green alga Agmenellum quadruplicatum. A preliminary report of these results has been presented (4). MATERIALS AND METHODSDNA purification. A. quadruplicatum strain PR-6 was cultivated axenically in medium A with NaNO3 (1 mg/ml) as described (5). Cells were harvested before reaching a density of 5 x 107 cells per ml, washed in 10% sucrose/50 mM Tris HCl, pH 8.0/100 mM Na2EDTA, and stored at -80°C. Lysis was achieved by thawing, adding egg-white lysozyme to a final concentration of 10 mg/ml, incubating at 37°C for 30 min, and adding N-lauroyl sarcosine (10% wt/vol stock solution) to a final concentration of 1%. An equal amount (wt/vol) of CsCl was added and DNA was purified by buoyant-density centrifugation. DNA-containing fractions were recentrifuged in the presence of ethidium bromide at 150 ,ug/ml. The dye was removed by n-butanol extraction and the DNA was dialyzed against 10 mM Tris-HCl, pH 8.0/1 mM Na2EDTA.RNA Purification. An exponentially growing culture of A. quadruplicatum was harvested, resuspended in the original volume of fresh medium A lacking nitrate, and incubated with aeration and illumination as before. The A620/A680 ratio decreased from 0.9 to 0.3 within 24 hr, whereupon the cells were harvested, resuspended in medium A with nitrate (1 mg/ml) and incubated as before. After 8 hr, cells were harvested by centrifugation and resuspended in 10 mM Tris HCl, pH 8.0/1 mM Na2EDTA. Cells were lysed by passage through a French pressu...
The lack of any known transduction or indigenous conjugation systems has left transformation as the major means for genetic manipulations in cyanobacteria. Studies of transformation in cyanobacteria generally have dealt with one of two distinct areas. The first area is genomic transformation where internalized donor DNA recombines with chromosomally located genes. Chromosomal transformation can be a powerful tool for genetic mapping and mutagenesis. The second area is plasmid transformation where internalized plasmid donor DNA becomes established as an independent replicon in the recipient cyanobacterium. This second area has received a great deal of attention because it allows the generation of merodiploids for studies of genetic regulation and control and because it potentially allows the expression of foreign genes in an oxygenic photoautotroph. This article will attempt to describe the development of our current understanding of these two types of genetic transformation in cyanobacteria.
Iacobucci (2009Iacobucci ( , 2010) covers a number of important issues in the use of structural equation modeling and in so doing provides researchers with many useful insights and sensible suggestions. This commentary focuses on three issues where our views differ somewhat from those expressed in the target articles: SEM and causal inferences, sample size, and model fit. In addressing each of these issues, our perspectives do not so much contradict the views expressed by Iacobucci as they reflect a somewhat different conceptual emphasis.There is little doubt that structural equation modeling (SEM) has been among the most influential and widely used statistical methods to emerge in consumer psychology and related disciplines over the past 30 years. As with any popular statistical method, applications of SEM in consumer psychology and other disciplines have not always followed the best available practices as suggested by the methodological literature. One reason for this gap between the methodological and substantive literatures is that many important methodological findings, by virtue of where they are published and their highly technical nature, are not readily accessible to the typical researcher. In light of this reality, articles such as those by Iacobucci (2009Iacobucci ( , 2010 provide a valuable resource to researchers.Our goal in the present commentary is not to reiterate the many useful insights made in the target articles or to echo the numerous sensible suggestions provided by Iacobucci. Instead, we have chosen to focus our comments on some select issues where our views differ somewhat from those expressed in the articles. In addressing these issues, it is worth noting that our views do not so much contradict what is expressed in the target articles, but rather reflect a somewhat different emphasis and conceptual perspective. SEM, the nature of data, and causal inferencesAs Iacobucci notes, SEM is most often utilized in the context of non-experimental data. Despite this fact, users of SEM often interpret their results in strong causal terms. Indeed, when SEM was a comparatively new technique in the social sciences, much of the initial enthusiasm for it seemed to be driven by the erroneous belief that it could allow researchers to miraculously transcend the inferential limitations of non-experimental data. Several decades of experience have made such extreme views less common, but it is difficult to dispute that the problem to some degree remains. This criticism notwithstanding, we think several additional observations regarding SEM and causal inferences are worth noting.First, in the strict sense of the term, we do not object to the use of the term "causal modeling" when referring to applications of SEM. Applications of SEM usually do involve the specification of models that make causal assumptions. Hence, researchers are modeling causal relations among variables. However, modeling a causal relation is not the same as proving a causal relation. Other models making different causal assumptions might pr...
Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb+ control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.
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