Hox gene duplication in fish.

The study of Hox gene clusters continues to serve as a paradigm for those interested in vertebrate genome evolution. Recent exciting discoveries about Hox gene composition in fishes challenges conventional views about vertebrate Hox gene evolution, and has initiated lively debates concerning the evolutionary events making the divergence of the major vertebrate lineages. Comparative analyses indicate that Hox cluster duplications occurred in early vertebrate evolution, and again within the order Cypriniformes of teleost fish. Loss of Hox genes was more widespread than duplication during fish evolution.

Genomic organization of the Hoxa4-Hoxa10 region from Morone saxatilis: implications for Hox gene evolution among vertebrates.

The physical mapping of Hox gene clusters from a limited number of vertebrates has shown an overall conservation in gene organization in which major evolutionary changes appear to be primarily restricted to the deletion of one or more genes, with the exception of the amplification of additional clusters as postulated from zebrafish. We have sequenced a 31 kb region of the HoxA cluster from the teleost Morone saxatilis (striped bass), both to provide a detailed physical map of this region and to better understand the nature of Hox cluster evolution among vertebrate taxa. We identified five linked Hox genes: Hoxa4, Hoxa5, Hoxa7, Hoxa9, and Hoxa10, which are organized similarly to those of other vertebrates. Furthermore, we have documented the absence of the Hoxa6 and Hoxa8 genes within the 31 kb contig. Comparison of our results to those published for other vertebrates suggests that the absence of Hoxa6 is a common characteristic of teleosts, whereas the absence of Hoxa8 is common to vertebrates in general, with the possible exception of zebrafish. Further comparisons between the HoxA genes from Morone with those from the pufferfish, Fugu rubripes, revealed the likely presence of a previously unreported Hoxa7 gene, or gene fragment, in the Fugu genome, which suggests that the Hoxa7 gene, unlike Hoxa6 or Hoxa8, is present in teleosts. In addition to these differences in vertebrate Hox cluster structure, we also observed a marked reduction in the length of the Hoxa4--a10 region between vertebrate lineages representative of teleosts and mammals. Comparative analysis of HoxA cluster organization among teleosts and mammals suggests that cluster length reduction and lineage-specific gene loss events are hallmarks of Hox cluster evolution.

Evaluation of the effects of fragmented steam exposure cycles on the survival of bacterial spores.

The purpose of this study was to examine the population and resistance characteristics of bacterial spores which have been exposed to an abbreviated steam sterilization cycle. The philosophy of many pharmaceutical manufacturers is to require a second complete terminal sterilization cycle in the event of an unplanned interruption during the terminal sterilization of a production batch. The impact of abbreviated steam sterilization cycles was examined for their effect on the survivability and resistance of bacterial spores following an inadequate sterilization cycle. Steam sterilization cycles of two minutes and four minutes were performed on separate groups of Biological Indicator spore strips. These groups were then held at room temperature and re-exposed to a range of sterilization conditions after 24, 48, and 72 hours, i.e., start cycle, abort, hold, start cycle, abort. Spore survivor curves were calculated and resistance estimations were determined. The results of the study indicated that the log level of the surviving spores remained fairly constant, but variability within groups increased as sterilization time increased. The resistance of these surviving spores, as measured by D value, also remained relatively constant throughout the holding period. Abbreviated cycles were similarly conducted on ampules containing a spore suspension, and the spore populations and moist heat resistances were determined over time. Contrary to the spore strip, the population of the subject ampules was less stable showing a gradual decline over the same observation period. The study also included a comparison of the surviving population of short and long fragmented cycles. The results of this study demonstrate that a second complete sterilization cycle is unnecessary to assure the absence of living matter in the sterilized units.

Characterization and Ecology of Carboxymethylcellulase-Producing Anaerobic Bacterial Communities Associated with the Intestinal Tract of the Pinfish, Lagodon rhomboides.

Carboxymethylcellulase (CMCase)-producing obligate anaerobes were isolated from the intestinal tract contents but not the feeding habitat of seagrass-consuming pinfish. Taxonomic characterization of these CMCase-producing strains revealed four taxonomic clusters; three were clostridial and one was of unknown taxonomic affinity. Our results demonstrated that the CMCase-producing obligate anaerobe community from pinfish differed from functionally similar microbial communities in terrestrial herbivores.

Survey of Hox-like genes in the teleost Morone saxatilis: implications for evolution of the Hox gene family.

We used a PCR-based method to survey a defined subset of Hox-like genes in the genome of Morone saxatilis (striped bass) to determine their relationship to similar Hox genes in other vertebrates, to estimate Hox cluster number, and to gain additional information about vertebrate Hox gene evolution. We identified eight distinct striped bass Hox-like genes representing Hox clusters A, B, and C. Comparison of Hox gene evolutionary trees from striped bass, zebrafish, and mouse provided evidence consistent with current views of early evolutionary divergence of genes from the Hox 3' and central classes. However, estimates of Hox gene divergence based on nucleotide substitution frequencies among four fish species and the mouse were contrary to established vertebrate phylogenies according to either traditional or molecular taxonomic analyses.

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.

Electrophoretic transfer of DNA, RNA and protein onto diazobenzyloxymethyl (DBM) - paper.

A method has been developed for the electrophoretic transfer of DNA, RNA, protein and ribonucleoprotein particles from a variety of gels onto diazobenzyloxymethyl (DBM) - paper. Conditions for the electrophoretic transfer of these macromolecules have been optimized to allow for nearly quantitative transfer and covalent coupling. DNA and RNA electrophoretically transferred to DBM-paper retain their ability to hybridize with specific probes. The high efficiency of transfer and the high capacity of DBM-paper for nucleic acids makes possible the sensitive detection of specific nucleotide sequences. Similar efficiency is achieved in electrophoretic transfer and covalent coupling of proteins to DBM-paper. Macromolecules can also be electrophoretically transferred and bound to DBM-paper incapable of covalent bond formation. Their elution from the paper in high salt provides a new and useful preparative method for isolation of DNA, RNA and protein.

7alpha-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.

The rate of 7alpha-dehydroxylation of primary bile acids was quantitatively measured radiochromatographically in anaerobically washed whole cell suspensions of Clostridium leptum. The pH optimum for the 7alpha-dehydroxylation of both cholic and chenodeoxycholic acid was 6.5-7.0. Substrate saturation curves were observed for the 7alpha-dehydroxylation of cholic and chenodeoxycholic acid. However, cholic acid whole cell K0.5 (0.37 micron) and V (0.20 mumol hr-1mg protein-1) values differed significantly from chenodeoxycholic acid whole cell K0.5 (0.18 micron) and V (0.50 mumol-1 hr-1 mg protein-1). 7alpha-Dehydroxylation activity was not detected using glycine and taurine-conjugated primary bile acids, ursodeoxycholic acid, cholic acid methyl ester, or hyocholic acid as substrates. Substrate competition experiments showed that cholic acid 7 alpha-dehydroxylation was reduced by increasing concentrations of chendeoxycholic acid; however, chenodeoxycholic acid 7alpha-dehydroxylation activity was unaffected by increasing concentrations of cholic acid. A 10-fold increase in cholic and 7alpha-dehydroxylation activity occurred during the transition from logarithmic to stationary phase growth whether cells were cultured in the presence or absence of sodium cholate. In the same culture, a similar increase in chenodeoxycholic acid 7alpha-dehydroxylation was detected only in cells cultured in the presence of sodium cholate. These results indicate the possible existence of two independent systems for 7alpha-dehydroxylation in C. Leptum.

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.

Characterization of 7-alpha-dehydroxylase in Clostridium leptum.

7-alpha-Dehydroxylation of primary bile acids was demonstrated radiochromatographically in whole cells of Clostridium leptum but was not observed in intestinal Bacteroides species. Activity of 7-alpha-Dehydroxylase was detected within a pH range of 5 to 9 and was 8-fold higher in specific activity in cell cultures in the presence of 0.1 mM sodium cholate than in its absence. 7-alpha-Dehydroxylase activity in whole cells was markedly inhibitied by 2,4-dinitrophenol, carbonyl-cyanide-m-chlorophenylhydrazine, and dicyclohexylcarbodiimide. A hypothesis concerning the dietary regulation of 7-alpha-dehydroxylating intestinal anaerobic bacteria is presented.

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.