Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.     

Gas chromatography/mass spectrometry method to quantify blood hydroxycitrate concentration

Hydroxycitrate (HCA), a popular dietary supplement for weight loss, is a competitive inhibitor of ATP-citrate lyase, an extramitochondrial enzyme involved in the initial steps of de novo lipogenesis (DNL). Although animal studies have shown that HCA effectively inhibits DNL and induces weight loss, these findings have not been consistent in humans. This raises the possibility that the bioavailability of HCA may differ among species. We developed a new GC/MS method to measure HCA levels in blood, using [U-(13)C]citrate (CA*) as internal standard to account for losses associated with the isolation, derivatization, and measurement of HCA. HCA and CA* were derivatized with BSTFA + 10% TMCS and analyzed using PCI/GC/MS (CA*, m/z 471; and HCA, m/z 553). The plasma HCA concentration was measured over a 3.5-h period in four subjects having ingested 2 g of HCA. Their plasma HCA concentration ranged from 0.8 to 8.4 microg/ml 30 min and 2 h after ingestion, respectively. These results demonstrate that when taken acutely, HCA is absorbed, yet present in small quantities in human plasma. This simple method requiring minimal sample preparation is able to measure trace amounts of HCA with accuracy and precision.     

Intragenic Suppression of a Capsid Assembly-Defective P22 Tailspike Mutation

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.

SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.     

Inter-specific differences in photosynthetic carbon uptake, photosynthate partitioning and extracellular organic carbon release by deep-water characean algae

1. The effect of light intensity on photosynthesis and the fate of newly fixed organic carbon was compared for three characean algae collected at the same depth (10 m) but differing in their depth distributions. For each species we determined photosynthesis–irradiance (P–E) responses, the partitioning of newly fixed carbon into four intracellular pools (low molecular‐weight compounds, polysaccharides, lipids and proteins) and the extracellular organic carbon (EOC) release at a range of photon flux densities (PFD) 0–60 μmol m^–2 s^–1. 2. The P–E responses differed between the three species, with the light compensation point (E_c) and dark respiration rate highest in the shallowest species (Chara fibrosa), intermediate in the mid‐range species (C. globularis) and lowest in the deepest species (C. corallina). Photosynthetic efficiency (α) and photosynthesis: respiration ratios were lowest in C. fibrosa and highest in C. corallina. 3. In all three species, the low molecular weight pool was the principal photosynthetic product (>60% of fixed C) at 3 μmol m^–2 s^–1 PFD, but its proportional contribution decreased rapidly with increasing irradiance. Polysaccharide rose to become the major product (>35% of fixed C) at saturating PFD (35 μmol m^–2 s^–1). 4. Protein synthesis was saturated at 5 μmol m^–2 s^–1 in all species and was consistently a lower proportion of the fixed carbon in C. corallina than the other species. The fraction incorporated in the lipid pool increased slightly with irradiance but was always less than 10% of fixed C, while the proportion lost as EOC was unaffected by light, being significantly higher in C. fibrosa than the other species. 5. A kinetic experiment with C. fibrosa at 35 μmol m^–2 s^–1 PFD revealed a continued increase in net polysaccharide, protein and lipid synthesis during a 22.5‐h light period, whereas the net size of the low molecular weight pool remained constant. In a subsequent dark period, protein and lipid synthesis continued at the expense of the polysaccharide and low‐molecular‐weight pools. The EOC release rose to a constant low release in the light, then peaked slightly immediately after the dark–light transition before returning to the same rate as in the light. Extrapolating these data over 24 h suggests that the proportion of fixed carbon lost as EOC may be as high as 10% in this species. 6. The interspecific differences in carbon acquisition between the three species reflected their depth distributions, with the deeper species having more efficient photosynthetic metabolism, lower P:R ratios and less EOC release, although no apparent differences in internal partitioning of photosynthate.     

ABSORPTION AND UTILIZATION OF IRRADIANCE BY CYANOBACTERIAL MATS IN TWO ICE-COVERED ANTARCTIC LAKES WITH CONTRASTING LIGHT CLIMATES

We investigated the under-ice light climate and the efficiency with which light was absorbed and utilized by benthic algal mats in Lakes Hoare and Vanda, two perennially ice-covered lakes in the McMurdo Dry Valleys area of Southern Victoria Land, Antarctica. The ice cover and water column of Lake Vanda were much more transparent than those of Lake Hoare (18% vs. 2% transmission though ice and attenuation coefficients for downwelling irradiance of 0.05 vs. 0.12 m ^{− 1}, respectively). In both lakes the under-ice spectra were dominated by blue-green wavelengths. The benthic flora under perennial ice covers of both lakes comprised thick mucilaginous mats, dominated by cyanobacteria. The mats were well suited to absorb the dominant blue-green wavelengths of the under-ice light, with phycoerythrin being present at high concentrations. The pigment systems of the benthic mats absorbed 30%–50% of the light that reached them, varying with depth and lake. There was a tendency for the percentage of absorption to increase as ambient irradiance decreased. The efficiency of utilization of absorbed irradiance was examined by constructing absorbed irradiance/oxygen evolution curves to estimate community quantum yield. Mats from 13 m in Lake Hoare showed the highest quantum yields, approaching 1 mol of carbon fixed for every 8 mol quanta absorbed under light-limiting conditions. Lake Vanda mats had lower quantum yields, but these increased with depth. Calculated in situ irradiance occasionally exceeded the measured saturating irradiance for oxygen evolution in both lakes, thus efficiency in situ was below the maximum at times. As in other environments, optimization strategies allowed efficient capture and utilization of the lower and middle ranges of experienced irradiance but led to a compromised capacity to use the highest irradiances encountered at each depth.