Search

We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70‐like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

Hepatocytes isolated from livers of fasted rats form little glycogen from glucose or lactate at concentrations below 20 mM. Glycogen is formed in substantial quantities at a glucose concentration of 60 mM. In the presence of 10 mM glucose, 20-30% as much glycogen as glucose is formed from fructose, sorbitol, or dihydroxyacetone. The addition of either glutamine, alanine, or asparagine stimulates the formation of glycogen from lactate 10- to 40-fold. The formation of glucose and glycogen is then about equal, and glycogen deposition in hepatocytes is similar to rates attained in vivo after fasted rats are refed. The amino acids stimulate 1.5- to 2-fold glycogen synthesis from fructose, and 2- to 4-fold synthesis from dihyDROXYACETONE. Ammonium chloride is about one-half as effective as amino acids in stimulating glycogen synthesis when glucose with lactate are substrates. It increased glycogen synthesis 25-50% from fructose but inhibited synthesis from dihydroxyacetone plus glucose.

We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70‐like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The ‘apparent turnover’ of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.

This study examines the structural relationship of mitochondria and the endoplasmic reticulum in liver. Livers of rat and Japanese quail were homogenized and fractionated in media of 0.25 M-sucrose, either 5mM or 50 mM in sodium Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], pH 7.4 (2.2 mM or 22 mM in Na respectively), designated here as low- and high-salt media. Three particulate fractions were prepared by sequential centrifugation. A nuclear pellet sedimenting at 300 g was obtained as described by Shore & Tata [(1977) J. Cell Biol. 72, 714-725], and from the resulting supernatant thereof a low-speed pellet (1100-1500 g) and a high-speed pellet (8000-10 000 g) were prepared. In the low-salt medium the yields of mitochondrial matrix enzymes (citrate synthase, glutamate dehydrogenase, ornithine carbamoyltransferase) and their specific activities in the low-speed pellet were over twice those in the high-speed pellet. In the high-salt medium the yield of matrix enzymes was 4-5 times, and the specific activities were up to 3 times, higher in the low-speed pellet than in the high-speed pellet. Oxygen uptake and respiratory control ratio were also much higher in the low-speed pellets in both media. Some 50-65% of the microsomal marker enzyme glucose 6-phosphatase was in the supernatant from the high-speed pellet, and the rest sedimented with the mitochondria. Repeated washing with the high-salt medium removes only a limited amount of reticulum. Washing with salt-free sucrose removes most of the reticulum, but a fraction remains strongly bound to mitochondria. Homogenates from quail and rat liver were fractioned isopycnically on Percoll gradients in either 0.25 M-sucrose or 0.25 M-sucrose/50 mM-sodium Hepes. Up to five particulate bands were separated and assayed. Mitochondria were present in two to three bands and were associated with endoplasmic reticulum. As seen in the phase-contrast microscope the mitochondria prepared in the low-salt medium consist of separate organelles. In the high-salt medium the mitochondria appear as chains of from three to ten organelles not touching each other. On addition of univalent ions at concentrations above 20 mM, the mitochondria aggregate into chains, and at higher ionic strength larger multidimensional aggregates are formed. The dispersion and aggregation of mitochondria are reversible. Negatively stained electron micrographs reveal a branched mitochondrial structure, with mitochondria held together by strands of reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Methods that use bacterial luciferase for the assay of NADH in the range from 1 pmol to 1 nmol are described. Optimal conditions for the assay of glycolytic intermediates, tricarboxylic acid-cycle intermediates and related amino acids from milligram amounts of tissue are presented. The whole spectrum of these intermediates can be determined on about 10 mg of liver tissue. The methods are simple, are suitable for routine use, and the instrumentation is inexpensive. The concentrations of glycolytic intermediates in rat livers were determined by conventional spectrometric methods and with luciferase, and the results found to be in good agreement.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12–15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50–60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.

The psbA genes, which encode the D1 protein of photosystem II, constitute a multigene family in the cyanobacterium Synechococcus sp. strain PCC 7942. Levels of messages from the three psbA genes change rapidly when cells are shifted from low-light to high-light conditions: the psbAI message level drops, whereas psbAII and psbAIII message levels increase dramatically. We examined the potential contributions of transcriptional and posttranscriptional processes in these high-light responses by subjecting cells that had been grown in a turbidostat at a standard light intensity (130 microeinsteins [microE] m-2 s-1) to either the same or a higher light intensity (500 microE m-2 s-1) in the presence or absence of rifampin. Northern (RNA blot) analysis of RNA isolated from cells subjected to high light showed that the increases in psbAII and psbAIII transcripts were blocked by rifampin. This suggests a transcriptional induction of these genes at high light intensities. Increased mRNA stability does not contribute to their accumulation in high-light conditions, since their half-life values did not increase relative to the half-lives measured at the standard light intensity. The rate of disappearance of the psbAI transcript in cells shifted to high light was diminished when either transcription or translation was blocked by rifampin or chloramphenicol, suggesting that accelerated degradation of the message requires de novo synthesis of a protein factor. When rifampin was added 10 min after the shift to high light intensity rather than before the shift, psbAI and psbAIII messages, but not the psbAII message, decayed at a faster rate. Susceptibility of the psbAIII transcript to the high-light-induced factor was also demonstrated by addition of chloramphenicol prior to the shaft to high light. psbAIII transcript levels went up more than twofold higher in chloramphenicol-treated cells than in untreated cells, whereas psbAII transcript levels were affected by the inhibitor. These experiments provide evidence that either new or increased synthesis of a degradation factor which affects a subset of Synechococcus transcripts occurs in cells subjected to high light intensity.

The psbAmultigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAIIand psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZand psbAIII-lacZtranslational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAIis expressed highest at low light, and both psbAIIand psbAIIIare expressed highest at high light. Along a gradient of light intensity from 5 µE· m−2·s−1to 482 µE·m−2·s−1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 µE·m−2·s−1suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.

It has long been suspected that chloroplasts evolved after an endosymbiotic event involving a photosynthetic prokaryote, presumably a cyanobacterium, and a eukaryotic organism. Recent studies have provided strong evidence about the cyanobacterial nature of chloroplasts1. Since the discovery of prochlorophytes2, oxygen-evolving photosynthetic prokaryotes containing chlorophyll a and chlorophyll b and lacking phycobiliproteins, there has been speculation that these represent evolutionary intermediates between cyanobacteria and chloroplasts3. Prochloron sp., the first described prochlorophyte, proved difficult to work with because it is an obligate symbiont of marine ascidians3.Prochlorothrix hollandica, a recently isolated, freshwater filamentous prochlorophyte, is easily maintained in the laboratory3,4. Overall pigment composition and thylakoid membrane structure of P. hollandica suggest it has intermediate characteristics between cyanobacteria and the chloroplasts of higher plants3. The P. hollandica psbA genes, which encode the photosystem II thylakoid protein Dl, were cloned and sequenced and the sequences compared to those reported for cyanobacteria, a green alga, a liverwort, and several higher plants. The two psbA genes present in P. hollandica encode an identical amino-acid sequence. As in all chloroplast psbA genes, there is a seven amino-acid gap near the C terminus of the derived protein relative to the protein predicted by cyanobacterial genes5, suggesting that P. hollandica is part of the lineage that led to chloroplasts after a divergence from cyanobacteria. This hypothesis is also supported by phylogenetic analysis of derived Dl amino-acid sequences from psbA genes of thirteen taxa on the basis of parsimony.

We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem II protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAI message following light/dark entrainment supported the reporter data. The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock.

Three psbA genes encoding the D1 protein of the photosystem II reaction center are differentially expressed under different light intensities in the cyanobacterium Synechococcus sp. strain PCC 7942. Two of the three psbA genes, psbAII and psbAIII, are induced rapidly when light intensity is increased from 125 x 10(-6) mol.m-2.s-1 to 750 x 10(-6) mol.m-2.s-1. A recombinational cloning vector that carries a transcriptional lacZ reporter gene was used to characterize the controlling elements responsible for light induction. At least three distinct cis elements are present in the regulatory regions of pbsAII and psbAIII: basal promoters, comparable to Escherichia coli sigma 70 promoters in position and sequence, confer constitutive expression of the genes under both low and high light intensities; negative elements upstream of the promoters down-regulate the expression of the corresponding gene; and sequences downstream of the promoters that correspond to the untranslated leader regions of the mRNAs (+1 to +41 in psbAII and +1 to +39 in psbAIII) are responsible for increased expression under high light. When these light-responsive elements were combined with an E. coli promoter (conII) in different positions and orientations, the expression of the lacZ gene was induced 4- to 11-fold. The induction of gene expression under high light by these enhancers was position independent but orientation dependent. When the elements were combined with the conII promoter in the correct orientation, they also conferred a small but reproducible level of light-responsive expression on this E. coli promoter.

The psbDI and psbDII genes in Synechococcus sp. strain PCC 7942 encode the D2 polypeptide, an essential component of the photosystem II reaction center. Previous studies have demonstrated that transcripts from psbDII, but not psbDI, increase in response to high light intensity. Soluble proteins from Synechococcus cells shifted to high light were found to have affinity for DNA sequences upstream from the psbDII coding region. DNA mobility-shift and copper-phenanthroline footprinting assays of a 258-bp fragment revealed three distinct DNA-protein complexes that mapped to the untranslated leader region between +11 and +84. Deletion of the upstream flanking region to -42 had no effect on the expression of a psbDII-lacZ reporter gene or its induction by light, whereas a promoterless construct supported only minimal background levels of beta-galactosidase. A 4-bp deletion within the first protected region of the footprint decreased the beta-galactosidase activity to approximately 2% of that of the undeleted control, but gene expression remained responsive to light. Deletion of the three protected regions completely abolished both gene expression and light induction. These results suggest that the psbDII gene requires elements within the untranslated leader region for efficient gene expression, one of which may be involved in regulation by light.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes which encode two forms of the D1 protein of photosystem II. Experiments using psbA-lacZ translational fusions and Western blot (immunoblot) analysis have shown that the psbA genes respond differently to changes in light intensity, altering the ratio of the two forms of D1 in the thylakoid membrane. Each gene produces a 1.2-kilobase (kb) mRNA. A probe specific for psbAII transcripts also identified a 1.6-kb mRNA which starts 419 base pairs upstream of the 5' end of the 1.2-kb species and overlaps the entire 1.2-kb transcript. This 419-base-pair region includes an open reading frame (ORF1) of 114 amino acids. We investigated the effects of changes in light intensity on psbAII transcript levels in a series of light shift experiments in the wild-type Synechococcus sp. and in AMC084, a mutant which does not produce the 1.6-kb transcript. After exposure to high light intensities for 15 min, the level of the 1.2-kb psbAII transcript increased in both strains. This transcript was not detected in either strain after transfer to low light intensity. The psbAIII transcript showed the same pattern of response as the 1.2-kb psbAII transcript, whereas the 1.6-kb psbAII transcript was unaffected by different light intensities. The psbAI transcript levels responded oppositely to those of psbAII and psbAIII. These data, considered along with previous results obtained by using lacZ translational gene fusions, indicate that the response of psbA genes to changes in light intensity is controlled primarily at the transcriptional level.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes encoding two forms of the D1 protein: form I, the product of psbAI, differs from form II, the product of both psbAII and psbAIII, at 25 of 360 residues. D1 is essential for photosynthesis as a core component of the photosystem II reaction center. Translational gene fusions between each of the Synechococcus psbA genes and the Escherichia coli lacZ gene were inserted into the chromosome of wild-type Synechococcus sp. at the respective psbA loci to serve as in vivo reporters of psbA expression. beta-Galactosidase activities indicated differential expression of the psbA-lacZ gene fusions related to light availability. Expression of psbAI was 500-fold greater than expression of psbAII and 50-fold greater than psbAIII under similar conditions. As light intensity decreased from 600 microE.m-2.s-1 to 2 microE.m-2.s-1, expression of the psbAI reporter increased eightfold while expression of the psbAII and psbAIII reporters decreased 10-fold, suggesting differential production of the two forms of D1 in photosystem II in response to light availability. Relative levels of psbA-lacZ fusion transcripts directly reflected beta-galactosidase activities in the transformants, although the fusion transcripts were less stable than native psbA messages.

When fasted rats ate regular lab chow there was a lag time of about 2 h before the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2) in liver began to rise from its low basal level. By contrast, in animals refed on a sucrose-based diet hepatic [Fru-2,6-P2] increased 20-fold (to a value of approximately 12 nmol/g wet weight) during the first hour. These responses correlated with differences in the ability of the two diets to increase the circulating [insulin]/[glucagon] ratio and thus to elevate the ratio of 6-phosphofructo-2-kinase to fructose-2, 6-bisphosphatase. Liver glycogen was deposited briskly in both groups of rats. To assess its mechanism of synthesis (directly from glucose versus indirectly via the gluconeogenic pathway), animals eating the chow or sucrose diets received intravenous infusions of [14C]bicarbonate, [1-14C] fructose, and 3H2O. After isolation, the glycogen was subjected to positional isotopic analysis of its glucose residues. The results established that regardless of the diet the bulk of liver glycogen was gluconeogenic in origin. The fact that with sucrose feeding carbon flow through hepatic fructose-1,6-bisphosphatase remained active despite high levels of Fru-2,6-P2 (a potent inhibitor of this enzyme in vitro) presents a metabolic paradox. Conceivably, the suppressive effect of Fru-2, 6-P2 on hepatic fructose-1,6-bisphosphatase is overridden in vivo by some unknown factor or factors generated in response to sucrose feeding. Alternatively, metabolic zonation in liver might result in the coexistence of hepatocytes rich in Fru-2,6-P2 (high glycolytic, low gluconeogenic, low glycogenic capacitites) with cells depleted of Fru-2,6-P2 (low glycolytic, high gluconeogenic, high glycogenic capacities).

The cyanobacterium Synechococcus sp. strain PCC 7942 has two copies of the psbD gene which encodes the D2 polypeptide of the photosystem II (PSII) reaction center. One of the genes, psbDI, overlaps the open reading frame of another photosystem II gene, psbC; the psbDII gene is monocistronic. Gene inactivation experiments had previously shown that psbDII is dispensable under normal laboratory growth conditions. However, similar experiments with psbDI never produced viable psbDI-inactivated mutants, presumably because psbC expression depends on transcription through psbDI. The experiments described here were designed to assess the need for psbDI independent of the need for expression of psbC. A strain, AMC027, was engineered in which a second copy of psbC was expressed from the psbDII locus. Northern (RNA) blot analysis confirmed that both psbDI and psbDII gave rise to dicistronic messages containing psbC sequences in AMC027. In this genetic background, it was possible to inactivate psbDI, creating strain AMC050 and indicating that the psbDII gene is functional. Western immunoblot analysis showed that the products of psbD and psbC, the PSII proteins D2 and CP43, respectively, were present in thylakoids of AMC050, but at reduced levels relative to the wild type, the mutant AMC027, and two psbDII-inactivated mutants. AMC050 consistently formed small colonies on plates and competed poorly in mixed-culture experiments. This suggested that, although not essential for viability, expression from the psbDI locus is required to produce sufficient D2 and CP43 for optimal growth.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.

Hepatocytes prepared from streptozotocin- and alloxan-diabetic rats starved for 24 h contain 0.5–2% wet wt. of glycogen. Glycogen synthesis in the hepatocytes from such rats, after prior depletion of the glycogen by glucagon injection, was studied. As distinct from cells from normal animals, there was no glycogen synthesis from glucose as sole substrate, even at concentrations of 60 mM. When supplied with glucose, a gluconeogenic precursor (lactate, dihydroxyacetone or fructose), and with glutamine there was concurrent synthesis of glucose and of glycogen. Without glutamine there was little or no glycogen synthesis. The rate of glycogen formation was in the same range as for cells from control rats. Glutamine addition markedly activated glycogen synthase in cells of starved diabetic rats, but there was no effect on phosphorylase. We obtained very little synthesis of glycogen with hepatocytes from fed diabetic rats, whereas with normal animals, synthesis by such cells equals or exceeds that obtained from starved rats. The conversion of synthase b (inactive) into the active form was studied in rat liver homogenates. The activation of the synthase in cells from starved diabetic rats is somewhat less than that from normal animals, but that from fed diabetic rats is markedly decreased compared with that in livers of fed control animals or that of starved diabetic animals.

Abstract: We designed a strategy to isolate and characterize response regulator genes from the cyanobacterium Synechococcus sp. strain PCC 7942 based on the premise that cyanobacterial response regulators would bear strong similarity to their counterparts from other eubacteria. Two response regulator genes, srrA and srrB, were isolated from Synechococcus and found to encode proteins similar to the OmpR subclass of response regulators. Disruption of either gene by insertional mutagenesis did not produce an obvious phenotype and did not affect the accumulation of psbAII mRNA under high-light conditions, indicating that these gene products are not involved in mediating the well characterized standard- to high-light transition response of photosystem II genes in this cyanobacterium. Analysis of the chromosomal region adjacent to srrA revealed the presence of another presumptive transcriptional activator gene. This gene, named lrrA, belongs to the lysR family. Attempts to disrupt lrrA or an adjacent ORF (orfG) were not successful, suggesting that these genes are important for the growth of Synechococcus.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.