SAAS bulletin, biochemistry and biotechnology最新文献

Transposon Tn1545 was introduced into the chromosome of the ruminal cellulolytic bacterium Eubacterium cellulosolvens. This was achieved by conjugal transfer of the transposon from Clostridium beijerinckii at a frequency of about 1 per 10(4) recipient cells. Transconjugants of E. cellulosolvens were resistant to both tetracycline and erythromycin. E. cellulosolvens could also serve as a donor for conjugal transfer of Tn1545 back into C. beijerinckii.

Our laboratory is engaged in an effort to identify genes expressed primarily during plant embryogenesis. Genes which exhibit unique expression profiles in the plant are also being sought. To this end, several methods to identify and clone novel genes based on specific expression patterns have been developed. These methods include virtual subtraction, differential display and other PCR based technologies. In addition to this, a yeast one-hybrid approach has been established to identify transcription factors which regulate these genes. To date, this work has identified several novel genes.

Synechocystis 6803 is a cyanobacterium that carries out-oxygenic photosynthesis. We are interested in the introduction of mutations in the large extrinsic loop region of the CP43 protein of Photosystem II (PSII). CP43 appears to be required for the stable assembly of the PSII complex and also appears to play a role in photosynthetic oxygen evolution. Deletion of short segments of the large extrinsic loop results in mutants incapable of evolving oxygen. Alterations in psbC, the gene encoding CP43, are introduced into Synechocystis 6803 by transformation and homologous recombination. Specifically, plasmid constructs bearing the site-directed mutations are introduced into a deletion strain where the portion of the gene encoding the area of mutation has been deleted and replaced by a gene conferring antibiotic resistance. We have constructed a deletion strain of Synechocystis appropriate for the introduction of mutations in the large extrinsic loop of CP43 and have used it successfully to produce site-directed mutants.

Heavy-atom derivatives of PYRase proteins prepared in the past have been unsuitable for x-ray diffraction analysis. Thus, we propose utilizing unnatural metalloid-containing amino acids as an alternative to heavy-atom derivatization. Selenomethionine-containing proteins analyzed by multiwavelength anomalous diffraction provides a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray Crystallography [Hendrickson, et al., 1991 and references therein]. Telluromethionine-containing proteins offer the same investigational potential, and additionally allow further simplification of the data collection technique by requiring only traditional methods of phase analysis [Boles et al., 1995 and references therein]. We sought to introduce the required Se and Te atoms into Staphylococcus aureus Pyrrolidone Carboxyl Peptidase (PYRase) via selenomethionine (SeMet) and telluromethionine (TeMet). Complete incorporation of SeMet into S. aureus PYRase was succeeded with little change in enzymatic properties. Incomplete incorporation (75%) of TeMet was accomplished in preparing TeMet-PYRase, however, representing the highest incorporation to date of a tellurium-containing amino acid. Enzymatic properties remained unchanged when TeMet was incorporated. We report herein the biosynthetic substitution and expression, protein purification and comparative biochemistry of SeMet-PYRase and TeMet-PYRase.

Dipyridamole (37.5 mg/kg, s.c., b.i.d.), a potent inhibitor of nucleoside transport, was administered to guinea pigs for 14 days in order to investigate the effects of: 1) chronic dipyridamole treatment on [3H]nitrobenzylthioinosine ([3H]NBMPR) binding: 2) chronically released endogenous adenosine on adenosine A1 and A2 receptors. Comparisons of the binding capacities (Bmax) and equilibrium dissociation constants (Kd) in vehicle-treated (VTA) and dipyridamole-treated animals (DTA), revealed a 100 percent increase in Kd of [3H]NBMPR binding in the kidney of DTA but not in heart or brain. There were no changes in adenosine A1 or A2 receptor activities in kidney and brain as measured by [3H]R-phenylisopropyladenosine and [3H]5'-N-ethyl-carboxamidoadenosine binding, respectively. The data suggest that cardiac and central nucleoside transporters may be either less susceptible to chronic dipyridamole administration or have a different adaptive mechanism. Also, endogenous adenosine, which may be chronically released upon dipyridamole treatment, has no effect on adenosine receptors.

Bombyxin, an insect hormone structurally related to insulin, was chemically synthesized and defined radioactively labeled probes were generated in order to detect and characterize bombyxin receptors in insect tissue. In all species tested, Bombyx mori (silkmoth), Samia cynthia ricini (Ailanthus moth). Manduca sexta (tobacco hornworm) and Spodoptera frugiperda (fall armyworm), bombyxin receptors were found in ovaries. A radioactive photolabeled bombyxin analog was crosslinked to the potential bombyxin receptor and the hormone-receptor complex identified by SDS PAGE. The bombyxin-receptor has an apparent molecular mass of about 300 kDa which after reduction forms a binding subunit of 90 to 110 kDa. The presence of bombyxin-receptors on ovaries suggests a function of this hormone in reproduction.

Saturating random mutagenesis at a given position within a polypeptide sequence can provide powerful insights into the functional requirements of the position. By coupling this genetic methodology with expression of human proteins in yeast, we and others have begun to ask pointed and important questions about the structure-function relationships of proteins associated with human genetic disease.

A 3.9 kb Haemophilus influenzae genomic DNA fragment was cloned in plasmid pUC9 that partially complemented the ultraviolet light sensitivity (UVs) of Escherichia coli uvrC mutant hosts. This fragment also complemented the UVs of H. influenzae uvr-1 (DB112) and uvr-2 (DB116) mutants. It genetically transformed the latter mutants to wild type UV resistance. The nucleotide (nt) sequence of this fragment revealed 3 open reading frames (ORFs). ORF2, now designated uvr-1+ (1746 nt), shows significant similarity in both the nt and amino acid (aa) composition to 7 complete proven or putative uvrC gene sequences. Computer analysis of the DNA sequence revealed several possible regulatory motifs 5' to uvr-1+, including a putative LexA-binding site as an inverted SOS box, located within the 3' region of ORF1, (extensive homology to the E. coli CMP-KDO synthetase gene), upstream of uvr-1+. Further computer analysis has also predicted that the four putative active site amino acids, located in the C-terminal half of each protein, are each situated in an area of secondary structure that are highly conserved.

The nucleoside transport system (NTS), an integral part of the adenosine inactivation mechanism, is physiologically significant in adenosine-mediated autoregulation of cardiovascular function. Radioreceptor approaches have been employed in the characterization of nucleoside transporters as potential targets in the therapy of cardiovascular diseases. Unanswered questions that are being addressed relate to their pharmacological regulation and their involvement in the pathogenesis of disease states. A brief overview of the characterization of nucleoside transporters in the cardiovascular system is presented and discussed in terms of their relevance to cardiovascular function.

Since we have previously shown that farnesol (F-OH) and geranylgeraniol (GG-OH) can be utilized for protein isoprenylation, the ability of the free allylic isoprenols to overcome the lovastatin-induced block on rat C6 glial cell proliferation has been investigated. Lovastatin an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in mevalonate biosynthesis, inhibited the rate of cell growth in a concentration dependent manner. The addition of mevalonate and GG-OH to lovastatin treated cells was shown to overcome the drug induced inhibition of cell proliferation. This result indicates that geranylgeraniol is capable of providing the mevalonic acid-derived products necessary to support cell growth. These results suggest that isoprenols are converted to an "activated' form, possibly the corresponding allylic pyrophosphate intermediate by reactions that remain to be characterized, prior to being utilized for sterol biosynthesis and protein isoprenylation.